Receptor gene screening for detecting or diagnosing cancer

ABSTRACT

Compositions and methods that use the body&#39;s natural secretory immune system in a new way against steroid hormone responsive tumors of the breast and prostate, as well as other glandular/mucus epithelial tissues such as colon, ovary, endometrium, kidney, bladder, stomach, pancreas and secretory pituitary gland are provided. Also provided are new ways of identifying carcinogenic, or potentially carcinogenic, bacteria in a tissue or body fluid to provide better anti-cancer therapies and preventatives than have been available previously.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application Nos. 60/203,314 filed May 10, 2000;60/208,348 filed May 31, 2000; 60/208,111 filed May 31, 2000; 60/229,071filed Aug. 30, 2000; and 60/231,273 filed Sep. 8, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Research leading to the present invention was supported in part by thefederal government under Grant Nos. DAMD17-94-J-4473, DAMD17-98-8337 andDAMD17-99-1-9405 awarded by the Defense Department through the US ArmyMedical Research and Materiel Command, Breast Cancer Research Program.The United States government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to risk assessment, detection,diagnosis, prognosis, treatment and prevention of steroid hormoneresponsive cancers of mucosal epithelial tissues (i.e., glands andtissues that secrete or are bathed by secretory immunoglobulins). Moreparticularly, the invention relates to negative (inhibitory) regulationof steroid hormone responsive cancer cell proliferation, and to theimmunoglobulin inhibitors and the receptors that mediate suchregulation.

2. Description of Related Art

Finding a naturally occurring biochemical defense mechanism capable ofcontrolling neoplastic growth has been the goal of a number ofresearchers for many years. Use of the immune system against malignanttumors forms the basis for many anti-cancer strategies. For example,U.S. Pat. No. 5,980,896 describes certain antibodies, antibody fragmentsand antibody conjugates and single-chain immunotoxins directed againsthuman carcinoma cells. Conventional anti-tumor immunotherapies rely onantibody-antigen recognition chemistry, and on targeting of antibodiesagainst various antigenic features of tumor cells in order to triggerdestruction of the tumor cells by the body's immune system or to targetthe tumor cells with antibody conjugates of various cytotoxic orchemotherapeutic agents. In practice, however, tumors in vivo havegenerally not been found to be very immunogenic and in many instancesappear to be capable of evading the body's immune response. Today agreat deal of anti-cancer work is directed at finding ways of increasingthe immunogenicity of a tumor cell in vivo. For example, U.S. Pat. No.6,120,763 (Fakhrai et al.) describes a method of preventing or reducingthe severity of a cancer in a subject by stimulating the subject'simmune response against the cancer. Many studies have attempted use ofIgG as passive immunity or stimulation of natural IgG production torestrict tumor growth. As of today, there are no known vaccines forbreast cancer, prostate cancer, or any other forms of mucosal cancers(Smyth M J et al. (2001) Nature Immunol 2, 293-299).

There is a second type of immune system that is very important to thefunction and protection of the body. The immunological function andphysiological properties of the body's secretory immune system has beenrecognized for many years (Tomasi T B et al. (1965) J Exp Med 121,101-124; Brandtzaeg P and Baklien K (1977) Ciba Foundation Symposium 46,77-113; Tomasi T B (1970) Ann Rev Med 21, 281-298; Spiegelberg H L(1974) Adv Immunol 19, 259-294; Tomasi T B (1976) The Immune System ofSecretions, Prentice-Hall, Englewood Cliffs, N.J.; Mestecky J and McGheeJ R (1987) Adv Immunol 40, 153-245). It was established thatimmunoglobulin A (IgA) represents 5 to 15% of the total plasmaimmunoglobulins in humans (Spiegelberg H L (1974) Adv Immunol 19,259-294). IgA has a typical immunoglobulin four-chain structure (M_(r)160,000) made up of two heavy chains (M_(r) 55,000) and two light chains(M_(r) 23,000) (Fallgreen-Gebauer E et al (1993) Biol Chem Hoppe-Seyler374, 1023-1028; Kratzin H et al. (1978) Hoppe-Seylers Z Physiol Chem359, 1717-1745; Yang C et al. (1979) Hoppe-Seylers Z Physiol Chem 360,1919-1940; Eiffert H et al. (1984) Hoppe-Seylers Z Physiol Chem 365,1489-1495). In humans, there are two subclasses of IgA. These are IgA1and IgA2 that have 1 and 2 heavy chains, respectively. The IgA2 subclasshas been further subdivided into A₂m(1) and A₂m(2) allotypes (Mestecky Jand Russell M W (1986) Monogr Allergy 19, 277-301; Morel A et al. (1973)Clin Exp Immunol 13, 521-528). IgA can occur as monomers, dimers,trimers or multimers (Lüllau E et al. (1996) J Biol Chem 271,16300-16309). In plasma, 10% of the total IgA is polymeric while theremaining 90% is monomeric. Formation of dimeric or multimeric IgArequires the participation of an elongated glycoprotein of approximatelyM_(r) 15,000 designated the “3” chain (Mestecky J et al. (1990) Am J Med88, 411-416; Mestecky J and McGhee J R (1987) Adv Immunol 40, 153-245;Cann G M et al. (1982) Proc Natl Acad Sci USA 79, 6656-6660).Structurally, the J chain is disulfide linked to the penultimatecysteine residue of heavy chains of two IgA monomers to form a dimericcomplex of approximately M_(r) 420,000. The general structure of thedimer has been well described in the literature (Fallgreen-Gebauer E etal (1993) Biol Chem Hoppe-Seyler 374, 1023-1028). Multimeric forms ofIgA and IgM require only a single J chain to form (Mestecky J and McGheeJ R (1987) Adv Immunol 40, 153-245; Chapus R M and Koshland M E (1974)Proc Natl Acad Sci USA 71, 657-661; Brewer J W et al. (1994) J Biol Chem269, 17338-17348). The structures and chemical properties of IgA and IgMhave been described in detail (Janeway C A Jr et al. (1996)Immunobiology, The Immune System in Health and Disease, Second edition,Garland Publishing, New York, pp 3-32 and pp 8-19).

Dimeric and multimeric IgA and IgM are secreted by a number of exocrinetissues. IgA is the predominant secretory immunoglobulin present incolostrum, saliva, tears, bronchial secretions, nasal mucosa, prostaticfluid, vaginal secretions, and mucous secretions from the smallintestine (Mestecky J et al. (1987) Adv Immunol 40, 153-245; Goldblum RM, et al. (1996) In: Stiehm E R, ed, Immunological Disorders in Infantsand Children, 4^(th) edition, Saunders, Philadelphia, pp 159-199;Heremans J F (1970) In: Immunoglobulins, Biological Aspects and ClinicalUses, Merler E, ed, National Academy of Sciences, Wash DC pp 52-73;Tomasi T B Jr (1971) In: Immunology, Current Knowledge of Basic Conceptsin Immunology and their Clinical Applications, Good R A and Fisher D W,eds, Sinauer Associates, Stanford, Conn., p 76; Brandtzaeg P (1971) ActaPath Microbiol Scand 79, 189-203). IgA output exceeds that of all otherimmunoglobulins, making it the major antibody produced by the body daily(Heremans J F (1974) In: The Antigens, Vol 2, Sela M, ed, AcademicPress, New York, pp 365-522; Conley M E et al. (1987) Ann Intern Med106, 892-899. IgA is the major immunoglobulin found in humanmilk/whey/colostrum (Ammann A J et al. (1966) Soc Exp Biol Med 122,1098-1113; Peitersen B et al. (1975) Acta Paediatr Scand 64, 709-717);Woodhouse L et al. (1988) Nutr Res 8, 853-864). IgM secretion is lessabundant but can increase to compensate for deficiencies in IgAsecretion. J chain containing IgA is produced and secreted by plasma Bimmunocytes located in the lamina propria just beneath the basementmembrane of exocrine cells (Brandtzaeg P (1985) Scan J Immunol 22,111-146). The secreted IgA binds to a M_(r) 100,000 poly-Ig receptorpositioned in the basolateral surface of most mucosal cells (Heremans JF (1970) In: Immunoglobulins, Biological Aspects and Clinical Uses,Merler E, ed, National Academy of Sciences, Wash DC, pp 52-73;Brandtzaeg P (1985) Clin Exp Immunol 44, 221-232; Goodman J W (1987) In:Basic and Clinical Immunology, Stites D P, Stobo J D and Wells J V, eds,Appleton and Lange, Norwalk, Conn., Chapter 4). The receptor-IgA complexis next translocated to the apical surface where IgA is secreted. Thebinding of dimeric IgA to the poly-Ig receptor is completely dependentupon the presence of a J chain (Brandtzaeg P (1985) Scan J Immunol 22,111-146; Brandtzaeg P and Prydz H (1984) Nature 311:71-73; Vaerman J-Pet al. (1998) Eur J Immunol 28, 171-182). Monomeric IgA will not bind tothe receptor. The J chain requirement for IgM binding to the poly-Igreceptor is also true for this immunoglobulin (Brandtzaeg P (1985) ScanJ Immunol 22, 111-146; Brandtzaeg P (1975) Immunology 29, 559-570;Norderhaug I N et al. (1999) Crit. Rev Immunol 19, 481-508). Because IgAand IgM bind to the poly-Ig receptor via their Fc domains, and becauseof a repeating Ig-like structure in the extracellular domains, thepoly-Ig receptor classifies as a member of the Fc superfamily ofimmungobulin receptors (Kraj{hacek over (c)}i P et al. (1992) Eur JImmunol 22, 2309-2315; Daëron M (1997) Annu Rev Immunol 15, 203-234).

During passage of IgA through the cell, its structure is modified. AM_(r) 80,000 fragment of the receptor containing all five of theextracellular domains becomes covalently attached to dimeric IgA to formsecretory IgA (sIgA) (Fallgreen-Gebauer E et al (1993) Biol ChemHoppe-Seyler 374, 1023-1028). The receptor that mediates thetranslocation has been interchangeably called the “poly-Ig receptor”(poly-Ig receptor) or the “secretory component” (Kraj{hacek over (c)}i Pet al. (1992) Eur J Immunol 22, 2309-2315). Except where notedotherwise, for the purposes of the present disclosure, the term “poly-Igreceptor” refers to the full length M_(r) 100,000 transmembrane proteinand the term “secretory component” denotes only the M_(r) 80,000extracellular five domains of the receptor that become covalentlyattached to IgA in forming the sIgA structure (Fallgreen-Gebauer E et al(1993) Biol Chem Hoppe-Seyler 374, 1023-1028; Kraj{hacek over (c)}i P etal. (1992) Eur J Immunol 22, 2309-2315). Because of the unique structureof sIgA, it is highly resistant to acid and proteolysis (Lindh E (1975)J Immunol 114, 284-286) and therefore remains intact in secretions toperform extracellular immunological functions. IgM also binds secretorycomponent, but not covalently (Lindh E and Bjork I (1976) Eur J Biochem62, 271-278). However, IgM is less stabilized because of its differentassociation with the secretory component, and therefore has a shorterfunctional survival time in acidic secretions (Haneberg B (1974) Scand JImmunol 3, 71-76; Haneberg B (1974) Scand J Immunol 3, 191-197). IgA andIgM are known to bind to bacterial, parasite and viral surface antigens.These complexes bind to receptors on inflammatory cells leading todestruction of the pathogen by antibody-dependent cell-mediatedcytotoxicity (Hamilton R G (1997) “Human immunoglobulins” In: Handbookof Human Immunology, Leffell M S et al., eds, CRC Press, Boca Raton,Chapter 3).

The major immunoglobulins secreted as mucosal immune protectors includeIgA, IgM and IgG. In human serum, the percent content of IgG, IgA andIgM are 80, 6 and 13%, respectively. In humans, the major subclasses ofIgG are IgG1, IgG2, IgG3 and IgG4. These are 66, 23, 7 and 4% of thetotal. IgG, respectively. The relative content of human immunoglobulinclasses/subclasses in adult serum follow the orderIgG1>IgG2>IgA1>IgM>IgG3>IgA2>IgD>IgE (Spiegelberg H L (1974) Adv Immunol19, 259-294). When the serum concentrations of immunoglobulins arecompared to those in exocrine secretion fluids, the relative contentschange dramatically (Brandtzaeg P (1983) Ann NY Acad Sci 409, 353-382;Brandtzaeg P (1985) Scand J Immunol 22, 111-146). For example incolostrum (a breast fluid secretion), IgA is ≧80% of the totalimmunoglobulins. IgM is ≦10% of the total. IgG represents a few percent.In human colostrum and milk, IgG1 and IgG2 are the major subclasses ofIgG (Kim K et al. (1992) Acta Paediatr 81, 113-118). Clearly, comparisonof serum and mucosal fluid concentrations indicate selectiveimmunoglobulin secretion. The secretion mechanism for IgA and IgM arewell described. Conversely, there is a fundamental question surroundingIgG secretion. There is no “J” chain present in IgG1 and IgG2. From theknown facts of transcytosis/secretion of immunoglobulins (Johansen F Eet al. (2000) Scand J Immunol 52, 240-248), it is unlikely that IgGsecretion is mediated by the poly-Ig receptor. An epithelial receptorspecific for IgG1 has been reported in bovine mammary gland (Kemler R etal. (1975) Eur J Immunol 5, 603-608). Apparently, it preferentiallytransports this class of immunoglobulins from serum into colostrum.Despite this 1975 report however, the receptor has not been chemicallyor structurally identified nor has the mechanism of transport of IgGmonomers been satisfactorily defined. Certainly no growth function wasascribed to this “IgG1 receptor” in the 1975 Kemler et al. report. It ispossible that this receptor is a member of a large group now designatedas Fc receptors (Fridman W H (1991) FASEB J 5, 2684-2690), but there isone study with IgG showing that of 31 different long-term humancarcinoma cell lines including breast “all lines were found to beconsistently Fc receptor negative” (Kerbel R S et al. (1997) Int JCancer 20, 673-679). One possible candidate for the epithelial transportof IgG1 is the neonatal Fc receptor (Raghavan M and Bjorkman P J (1996)Annu Rev Cell Dev Biol 12, 181-220). However, there is no indication yetof the presence of this receptor in adult mucosal tissues.

All human mucus membranes are protected by the secretory immune system(Hanson L Å and Brandtzaeg P (1989) In: Immunological Disorders inInfants and Children, 3^(rd) edition, Stiehm E R, ed, Saunders,Philadelphia, pp 169-172). The primary protector is sIgA that isproduced as dimers and larger polymers. A single joining “J” chainconnects IgA monomers to form the dimers and polymers (Garcia-Pardo A etal. (1981) J Biol Chem 256, 11734-11738), and connects monomers of IgMto give pentamers (Niles M J et al. (1995) Proc Natl Acad Sci USA 92,2884-2888). This critical joining endows these structures with a veryimportant immunological property. Dimeric and polymeric sIgA have a highantigen binding valence that effectively agglutinates/neutralizesbacteria and virus (Janeway C A Jr et al. (1999) Immunobiology, TheImmune System in Health and Disease, 4^(th) edition, Garland Publishing,New York, pp. 326-327). Also, sIgA shows little or no complementactivation. This means that it does not cause inflammatory responses(Johansen F E et al. (2000) Scand J Immunol 52, 240-248). In addition,the fact that IgA exists as two separate forms is significant (Loomes LM et al (1991) J Immunol Methods 141, 209-218). The IgA1 predominates inthe general circulation. In contrast, IgA2 is often higher in mucosalsecretions such as those from breast, gut, and respiratory epithelium,salivary and tear glands, the male and female reproductive tracts, andthe urinary tracts of both males and females. This difference inproportions is important to immune protection of mucosal surfaces.Although the secretory form of IgA1 is by and large resistant toproteolysis (Lindh E (1975) J Immunol 114, 284-286), a number ofdifferent bacteria secrete proteolytic enzymes that cleave it into Faband Fc fragments (Warm J H et al. (1996) Infect Immun 64, 3967-3974;Poulsen K et al. (1989) Infect Immun 57, 3097-3105; Gilbert J V et al.(1988) Infect Immun 56, 1961-1966; Reinholdt J et al. (1993) InfectImmun 61, 3998-4000; Blake M S and Eastby C (1991) J Immunol Methods144, 215-221; Burton J et al. (1988) J Med Chem 31, 1647-1651; MortensenS B and Kilian M (1984) Infect Immun 45, 550-557; Simpson D A et al.(1988) J Bacteriol 170, 1866-1873; Blake M S and Swanson J et al. (1978)Infect Immun 22, 350-358; Labib R S et al. (1978) Biochim Biophys Acta526, 547-559). In effect, the bacterial proteinases negate theneutralizing effects of multivalent sIgA1. In contrast, because ofstructural differences (Chintalacharuvu K R and Morrison S L (1996) JImmunol 157, 3443-3449), IgA2 lacks sites required for proteolysis. Thismakes IgA2 more resistant to bacterial digest than IgA1 (Hamilton R G(1997) “Human immunoglobulins” In: Handbook of Human Immunology, LeffellM S et al., eds, CRC Press, Boca Raton, Chapter 3). With regard to IgM,its function is somewhat different. IgM antibodies serve primarily asefficient agglutinating and cytolytic agents. They appear early in theresponse to infection and are largely confined to the bloodstream.Whether secreted or plasma-borne, IgM is a highly effective activator ofthe classical complement cascade. It is less effective as a neutralizingagent or an effector of opsinization (i.e. facilitation of phagocytosisof microorganisms). Nonetheless, IgM complement activation causes lysisof some bacteria. The effects of the IgG class are more encompassing.All four subclasses cause neutralization, opsinization and complementactivation to defend against mucosal microorganisms. IgG1 is an activesubclass in this regard (Janeway C A Jr et al. (1999) Immunobiology, TheImmune System in Health and Disease, 4^(th) edition, Garland Publishing,New York, pp 326-327).

With regard to breast cancer and prostate cancer etiology, there hasbeen only limited attention given to the role of the immune system.Other issues have been considered more important for placing individualsin the at-risk groups for developing cancer in general and breast cancerspecifically. This has led to searches for risk factors. Advancescertainly have been made. We now have the benefit of the investment ofscientific effort and volume of new information that was obtained.Breast cancer is one useful example of our advances. There have beenseveral reviews of this topic published since 1979 (Kelsey J L (1979)Epidemiol Rev 1, 74-109; Kelsey J L and Berkowitz G S (1988) Cancer Res48, 5615-5623; Kelsey J L and Gammon M D (1990) Epidemiol Rev 12,228-240; Colditz G A (1993) Cancer 71, 1480-1489; Alberg A J andHelzlsouer K J (1997) Current Opinion Oncology 9, 505-5111). Althoughreproductive factors, body build, oral contraceptives, estrogenreplacement therapy, diethylstilbestrol, hormonal imbalances, diet(particularly high fat consumption), alcohol consumption, radiation,familial aggregation and heredity have been studied, and some of theseidentified as risk factors, there remains no known cause of the 70% ormore of breast cancers now known as “sporadic” because they appear tooccur randomly in the population and certainly without any known geneticpattern. Plainly stated, for the vast majority of women who developbreast cancer, there is no known genetic cause. Even with the bestapplications of the epidemiology cited above, the answer has not beenforthcoming for this majority.

The only cases where there is a defined genetic origin of breast cancerinvolve the BRCA1 and BRCA2 genes. The BRCA1 gene has been cloned,sequenced and localized to chromosome 17 (Hall J M et al. (1990) Science(Wash DC) 250, 1684-1689; Bowcock A M (1993) Breast Cancer Res Treat 28,121-135; Mild Y et al. (1994) Science (Wash DC) 266, 66-71). Anothergene, BRCA2, has also been identified and linked to chromosome 13q(Wooster R et al. (1995) Nature (Lond) 378, 789-792; Tavigian S V et al.(1996) Nature Genet. 12, 333-337). BRCA1 gene lesions are linked tobreast and ovarian cancer. BRCA2 is more associated with ovarian cancerthan breast cancer. Together, these two genes are thought to account formost of the inheritable/familial breast cancer in the United States(Krainer M et al. (1997) New Eng J Med 336, 1416-1421). However, oneimportant fact that must be recognized is that these genes are probablycarried by fewer than 400 women in the United States and therefore areresponsible for a relatively small number of human breast cancers (KingM-C et al (1993) JAMA 269, 1975-1980; Biesecker B B et al. (1993) JAMA269, 1970-1974). Although these genes continue to be studiedintensively, it is far from clear that they have a significant causativerole in the 70% or more of “sporadic” non-inherited breast cancers. Infact, the essential point is that the origin of the vast majority ofbreast cancers remains unknown.

Currently these two genes, BRCA1 (Lynch H et al. (1978) Cancer 41,1543-1549; Hall J M et al. (1990) Science (Wash DC) 2500684-1689; NarodS A et al. (1991) Lancet 338, 82-83; Steichen-Gersdorf E et al. (1994)Am J Hum Genet. 55, 870-875; Mild Y et al. (1994) Science (Wash DC) 266,66-71; Smith S et al. (1992) Nature Genet. 2, 128-131) and BRCA2(Wooster R et al. (1994) Science (Wash DC) 265, 2088-2090; Wooster R etal. (1995) Nature 378, 789-792), have been related to early onset offamilial (autosomal dominant) breast and ovarian cancer. In contrast toBRCA1, which is linked predominantly to female cancers, BRCA2 is alsolinked to male breast cancer. As pointed out above, about 1% of thebreast cancers occurring in the United States are related to those genes(Easton F D et al. (1994) Lancet 344, 761). Their gene sequences havebeen fully characterized and in the case of BRCA1, many mutations havebeen identified (Shattuck-Eidens D et al. (1995) JAMA 273, 535-552;Simard J et al. (1994) Nature Genet. 8, 392-398; Castilla L H et al.(1994) Nature Genet. 8, 387-391). Mutations in these genes wereinitially considered to confer more than 80% lifetime risk fordeveloping breast and/or ovarian cancer (Easton D F et al. (1993) Am JHum Genet. 52, 678-701). More recent results have reduced the roles ofBRCA1 and BRCA2 in breast cancers (Struewing J P et al. (1997) New Eng JMed 336, 1401-1408; Couch F J et al. (1997) New Eng J Med 336,1409-1415; Krainer M et al. (1997) New Eng J Med 336, 1416-1421). BRCA1and BRCA2 may have roles in sporadic breast and ovarian cancers, but towhat extent is open to question (Futreal P A et al. (1994) Science (WashDC) 266, 120-122; Merajver S D et al. (1995) Nature Genet. 9, 439-443).In addition to BRCA1 and BRCA2, the tumor suppressor gene p53 has beenimplicated in both familial (germ line) and sporadic breast cancers(Malkin D et al. (1990) Science (Wash DC) 250, 1233-1238; Coles C et al.(1992) Cancer Res 52, 5291-5298; Elledge R M and Allred D C (1994)Breast Cancer Res Treat 32, 39-47). However, this genetic link accountsfor at most 25% of breast cancers. It is possible that germ linemutations in p53 also are related to a fraction of prostate cancers(Malkin D et al. (1990) Science (Wash DC) 250, 1233-1238). One area ofactive investigation focuses on the 70% of breast cancers termed“sporadic,” because they are not familial and not related to anycurrently known epidemiological risk factor. An effective means ofassessing genetic risk for sporadic breast cancers, prostate cancers,and other cancers of glandular/mucosal epithelial tissues, simply doesnot exist today in the conventional medical arsenal against cancer.

The genetic origin of prostate cancers has been even more elusive thanthat of breast cancers. Although a gene for prostate cancersusceptibility has been localized to chromosome 17q, it does not appearto be related to BRCA1 (PCT Pub. App. No. WO0027864). Other prostatecancer susceptibility genes have been localized to chromosomes 13q(Cooney K A et al. (1996) Cancer Res 56, 1142-1145) and to chromosomes8p, 10q and 16q (Veronese M L et al. (1996) Cancer Res 56, 728-732).From the data available, it is clear that the genetic origin of prostatecancer has not been identified. This fact alone opens the issue ofcause. While genetic analysis will continue to be important, it will notprovide the essential information about what is causing breast andprostate cancer.

In a conceptually different approach to identifying cancer-relatedgenes, Dr. Ruth Sager has suggested a departure from the conventionalavenues of identifying cancer-related genes by searching for mutations(Class I genes), and instead or additionally focusing on the role ofexpression genetics in cancer (Class II genes) (Sager R (1997) Proc NatlAcad Sci 94, 952-955). Dr. Sager has proposed that far more genes aredown regulated at the transcriptional level in cancer cells than aremutated and that crucial “oncogenic” molecules may not be mutated.Consistent with that proposition others have reported (Thompson M F etal. (1995) Nature Genet. 9, 444-450) that reduced amounts of BRCA1 mRNA,representing down-regulation of the wild-type gene, were found inprimary tumors of the nonfamilial disease. Characterization of othergenes whose expression is altered in cancer cells, and understandingtheir functions, will provide penetrating insight into the regulatoryinteractions that have been upset in cancer.

With regard to the origins of mucosal cancer, and especially breast andprostate, there has been little advance. In general, it is thought thatenvironmental carcinogens are the origin. However, this has yet to beproven. Another familiar concept is the idea that bacteria may beinvolved in carcinogenesis (oncogenesis). For example, see Parsonnet J(1995) Environ Health Perspect 103 (Suppl), 263-268; Mackowiak P A(1987) Am J Med 82, 79-97; Cassell G H (1998) Emerg Infect Dis 4,475-487; Nauts H C (1989) Cancer Surv 8, 713-723; Venitt S (1996)Environ Health Perspect 104 (Suppl), 633-637; Miller J H (1996) CancerSurv 28, 141-153; Buiuc D and Dorneanu O (1989) Rev Med Chir Soc Med NatIasi 93, 223-227).

Involvement of bacteria, or other infectious agents, in some types oflymphoid cancers such as Hodgkin's disease and leukemia has beensuggested (Comment of Editor: Infective cause of childhood leukaemia(1989) Lancet 1 (1829), 94-95; Serraino D et al. (1991) Int J Cancer 47,352-357; Glaser S L and Jarrett R F Baillieres (1996) Clin Haematol 9,401-416; Wolf J and Diehl V (1994) Ann Oncol 5 (Suppl 1), 105-111).

Studies suggesting that Helicobacter pylori is directly causative ingastric cancer have recently been described. H pylori is the onlybacterium known to date to have been classified as a Class I carcinogenby the International Agency for Research on Cancer (IARC). Thisclassification indicates that by generally accepted scientific standards(Nyren 0 (1998) Semin Cancer Biol 8, 275-283) this microorganism is nowgenerally considered to be a causative factor in development of gastriccancers in infected humans. Recently it has been reported that Chlamydiatrachomatis infection is strongly associated with subsequent developmentof invasive cervical squamous cell carcinoma (Anttila T et al. (2001)JAMA 283, 47-51). The possibility that bacteria are involved in largebowel/colon cancer has also been mentioned (McBurney M I et al. (1987)Nutr Cancer 10, 23-28), however no firm conclusions have been reached asyet.

Finally, the issue of prevention deserves special comment. There are noknown methods of preventing cancer other than observing life stylechanges and environmental changes that place individuals in the low riskgroups. Tamoxifen has been considered as a potential “prevention” forbreast cancer in high risk women, but as yet has not been widelyaccepted because of the physiologic and endocrine aberrations caused bythis agent when used long term. In short, even though prevention isremarkably pressing, there has been a dearth of studies of new methodsthat do not disrupt the normal lifestyles and reproductive capacity ofwomen.

Conventional immunological approaches to treating malignant tumors havegenerally proven inadequate. In addition, except for recent advanceswith respect to Helicobacter pylori and Chlamydia trachomatis (Anttila Tet al. (2001) JAMA 283:47-51), anti-bacterial approaches for combatingthe cause(s) of malignant transformation do not appear promising.Relying only on the existing technologies, effective diagnostic andtherapeutic agents, treatments and preventatives for widespread use inbreast and prostate cancers, and cancers of other glandular/mucosalepithelial tissues, do not appear to be on the near horizon.

SUMMARY OF THE INVENTION

New methods and compositions for use against steroid hormone responsivetumors of the breast and prostate, as well as against tumors of otherglandular/mucus epithelial tissues such as colon, ovary, endometrium,kidney, bladder, stomach, pancreas and secretory pituitary gland areprovided which are based on previously unrecognized activities ofcertain components of the body's natural immune system. References inthis disclosure to the “new natural immune mechanism” or the “newimmunotherapies,” refer to the previously unrecognized cell growthinhibitory function of certain constituent parts of the secretory immunesystem, particularly dimeric/polymeric IgA, polymeric IgM and IgG1, asdistinguished from the well known functions of those immunoglobulinsbased on antigen-antibody recognition. For the purposes of thisdisclosure, the term “cell growth” refers to cell proliferation or anincrease in the size of a population of cells rather than merely to anincrease in cytoplasmic volume of an individual cell. The term “steroidhormone responsive” cell refers to a cell that requires the binding of asteroid hormone to a steroid hormone binding receptor in the cell inorder for that cell to be stimulated to grow (i.e., proliferate). Forexample, normal ductile cells in the pubescent breast are estrogenresponsive or stimulated by estrogen to proliferate. ER⁺ breast cancercells also possess a functional estrogen binding receptor and are alsoestrogen responsive. By contrast, ER⁻ breast cancer cells do not have afunctional estrogen receptor and demonstrate autonomous cell growth,i.e., they are stimulated to proliferate without the influence of asteroid hormone. New ways of identifying carcinogenic, or potentiallycarcinogenic, bacteria in a tissue or body fluid are also provided, and,individually or together with the above-described newimmunotechnologies, are expected to provide or aid in widespreadimplementation of better anti-cancer therapies and preventatives thanhave been available previously.

In accordance with certain embodiments of the present invention, methodsof assessing risk or susceptibility of an individual to developing aneoplastic lesion or cancerous tumor of a mucosal epithelial tissue areprovided. In some embodiments the method includes detecting, and in somecases also quantitating, a steroid hormone reversible immunoglobulininhibitor of steroid hormone responsive cell growth in a body fluid orsecretion obtained from said subject, such as serum, plasma, colostrum,breast aspirates, saliva, tears, bronchial secretions, nasal mucosa,prostatic fluid, urine, semen or seminal fluid, vaginal secretions,ovarian aspirates, stool, and mucous secretions from the small intestineor stomach. The absence or deficiency of the immunoglobulin inhibitorcompared to a predetermined standard material indicates or suggests thata steroid hormone responsive mucosal epithelial tissue in the body ofthe individual is secreting or bathed by less than a cell growthinhibitory amount of the immunoglobulin inhibitor. For the purposes ofthis disclosure, the term “immunoglobulin inhibitor” refers to asecretory immunoglobulin, preferably one or more of the secretoryimmunoglobulins IgA, IgM and IgG1, that is active for inhibitingproliferation of a steroid hormone responsive cancer cell maintained ina suitable nutrient medium under cell growth promoting conditions, inthe absence of an inhibition-reversing amount of the steroid hormone orother substance that mimics this steroid hormone effect. Theimmunoglobulin inhibitory activity, also referred to as immunoglobulininhibition, is distinct from any additional antigen-antibody recognitionbased immunological functions of the immunoglobulin inhibitors. The term“steroid hormone reversible immunoglobulin inhibitor” refers to thecharacteristic of the preferred immunoglobulin inhibitors that theircell growth inhibitory activity is steroid hormone reversible. “Cellgrowth promoting conditions” refer to favorable environmentalconditions, other than defined medium components, and include suchthings as gaseous environment, humidity, temperature, pH, and the like.For example, cell growth promoting conditions could include incubationat 37° C. in a humid atmosphere of 5% (v/v) CO₂ and 95% (v/v) air in adefined nutrient medium at pH 7.4.

In certain embodiments the risk assessment method includes measuring theamount and/or activity of an immunoglobulin inhibitor in a specimencomprising a defined amount of body fluid or secretion from theindividual. In certain preferred embodiments the method includessubstantially depleting steroid hormone from the fluid specimen to yielda steroid hormone depleted specimen, and then assaying an aliquot ofthat hormone depleted specimen for detecting or measuring steroidhormone reversible inhibition of steroid hormone responsive cancer cellproliferation.

Some embodiments of the risk assessment method include the followingassay protocol: (a) maintaining a predetermined population of steroidhormone-responsive cells in a ferric ion-free, calcium ion-containing,serum-free nutrient medium, the cells being serum free and obtained froma steroid hormone-responsive cell line; (b) adding a predeterminedamount of the steroid hormone to the medium, the amount being sufficientto stimulate cell growth under cell growth promoting conditions; (c)adding a predetermined amount of a steroid hormone depleted specimen ofa body fluid or secretion to the medium, to yield a test mixture; (d)incubating the test mixture for a predetermined period of time undercell growth promoting conditions; (e) measuring the cell population inthe test mixture after the predetermined period of time; (f) measuringthe cell population in a control incubation mixture like the testmixture, except lacking an amount of the specimen. Preferably anycytotoxic effects of the specimen are also measured. The differencebetween the cell populations before and after the incubation period isdetermined, a significant increase in the population indicating theabsence of inhibition of cell growth by that amount of specimen in thepresence of the selected amount of steroid hormone. A significant lackof increase in the cell population, which is not attributable tocytotoxic effects of the specimen, is an indicator of inhibition of cellgrowth by the inhibitors contained in the specimen when tested in thepresence of a given concentration of steroid hormone. In preferredembodiments the assay also includes detecting or determining steroidhormone reversibility of the specimen's inhibitory activity in thepresence of a predetermined increased amount of steroid hormone.

Also provided in accordance with certain embodiments of the presentinvention are an in vitro method of, detecting loss of immunoglobulinregulation of steroid hormone responsive cell growth. In certainembodiments the method comprises assaying for inability of a mucosalepithelial cell to bind at least one of the immunoglobulins IgA, IgM andIgG1.

Certain other embodiments of the invention provide a method of detectinga mediator of immunoglobulin inhibition of steroid hormone responsivecell growth that includes detecting a poly-Ig receptor in a mucosalepithelial cell.

Certain other embodiments of the invention provide a method of detectinga gene coding for a mediator of immunoglobulin inhibition of steroidhormone responsive cell growth that includes detecting the presence of apoly-Ig receptor gene in a mucosal epithelial cell.

Still other embodiments of the invention provide a method of detecting agenetic defect in a gene coding for a mediator of immunoglobulininhibition of steroid hormone responsive cell growth comprisingscreening a genomic or cDNA library of a mucosal epithelial cell for adefect in a poly-Ig receptor gene.

Some embodiments of the present invention provide a method of detectingexpression of a defective mediator of immunoglobulin inhibition ofsteroid hormone responsive cell growth in a specimen of mucosalepithelial tissue, the method including detecting a defective poly-Igreceptor or Fcγ receptor protein in said specimen. The term “defective”means that the detected protein is physically similar to the nativereceptor protein, but is incapable or less capable of mediating theimmunoglobulin cell growth inhibitory effects, compared to the nativereceptor protein. Preferably the ability to mediate cell growthinhibitory effects is measured in a cell growth assay as describedelsewhere herein.

In accordance with certain other embodiments of the present invention,methods to aid in predicting increased susceptibility of a mammaliansubject to development or growth of a steroid hormone responsive cancerin a mucosal epithelial tissue is provided. In some embodiments, themethod comprises detecting the loss or impairment of negative regulationof breast tissue proliferation by the secretory immune system. In someembodiments, the method includes assaying a specimen of mucosalepithelial tissue obtained from the subject for the presence of apoly-Ig receptor capable of mediating immunoglobulin inhibition ofsteroid hormone responsive cell growth in a suitable in vitro cellculture assay. An absence of the receptor or absence of its activity formediating the immunoglobulin inhibition is suggestive that the tissuelacks functional mediators of immunoglobulin inhibition sufficient todeter development or growth of a steroid hormone responsive cancer ofthe mucosal epithelial tissue.

Also provided by certain embodiments of the invention is a method to aidin detecting transformation of a mucosal epithelial cell from normallysteroid hormone responsive to a steroid hormone responsive neoplastic,precancerous or cancerous condition, the method including assaying apopulation of the cells for loss or inactivity of receptors that mediateIgG1 inhibition of cell growth.

Further provided by certain embodiments of the invention are methods toaid in detecting progression of a steroid hormone responsive malignantmucosal epithelial cell to an autonomous cancer cell. In someembodiments, the method includes assaying for loss or inactivity of areceptor that mediates IgA and/or IgM inhibition of cell growth.

According to certain other embodiments of the invention, methods ofimaging a steroid hormone responsive mucosal epithelial tumor in vivo isprovided. In certain embodiments, the method includes contacting thetumor with at least one tagged or labeled monoclonal antibody raisedagainst a poly-Ig receptor, Fcγ receptor, IgA, IgM and IgG1, and thenimaging the tag.

In some embodiments of the present invention a method to aid indetecting or diagnosing cancer in a mammalian subject is provided. Themethod comprises determining in a population of neoplastic cells in amucosal epithelial tissue specimen obtained from the subject at leastone of the following conditions: (a) absence or diminution ofimmunoglobulin inhibition of steroid hormone responsive cell growth; (b)absence or diminution of at least one immunoglobulin inhibitor ofsteroid hormone responsive cell growth from a body fluid or secretionsecreted by or bathing said tissue; (c) absence or diminution of apoly-Ig receptor in said cells; (d) absence of a poly-Ig receptor genefrom said cells; (e) absence of heterozygosity for said poly-Ig receptorgene in said cells; (f) absence or diminution of a Fcγ receptor in saidcells; (g) absence of a Fcγ receptor gene from said cells; (h) absenceof heterozygosity for said Fcγ receptor gene in said cells; (i) absenceor diminution of TGFβ regulation of cell growth; (j) absence ordiminution of a TGFβ receptor in said cells; (k) absence of a TGFβreceptor gene from said cells; and (l) absence of heterozygosity forsaid TGFβ receptor gene in said cells. The absence or diminution ispreferably measured by comparison to similar determinations innon-neoplastic cells from the same patient or by comparison topredetermined standard values. The presence of at least one of thoseconditions is suggestive or indicative of the presence of a cancerous orprecancerous lesion, and an absence of one or more of the conditionssuggests or indicates absence of a cancerous or precancerous lesion inthe patient.

In accordance with certain additional embodiments of the invention, amethod to aid in staging a cancer of a mucosal epithelial tissue isprovided. The method includes testing or determining, in a specimen ofneoplastic cells obtained from the cancer, if the cells are stimulatedby a preselected steroid hormone proliferate in a suitable cell growthnutrient medium, and also determining at least one of the followingconditions: (a) in a specimen of body fluid or secretion secreted by orbathing said mucosal epithelial tissue, the lack of a cell growthinhibitory amount of at least one immunoglobulin inhibitor of steroidhormone responsive cell growth, (b) loss or diminution of a TGFβreceptor in said cells, (c) loss of a TGFβ receptor gene in said cellsin said cells, (d) loss of heterozygosity for said TGFβ receptor gene insaid cells, (e) loss or diminution of a poly-Ig receptor in said cells,(f) loss of a poly-Ig receptor gene in said cells, (g) loss ofheterozygosity for said poly-Ig receptor gene in said cells, (h) loss ordiminution of a Fcγ receptor in said cells, (i) loss of a Fcγ receptorgene in said cells, and (j) loss of heterozygosity for said Fcγ receptorgene in said cells. The loss or diminution in each case is measured bycomparison to similar determinations in non-neoplastic cells from thesame patient, or by previous values obtained from a previous test, or bycomparison to predetermined standard values. The presence of one or moreof the conditions suggests or indicates advancement of the stage of thecancer.

According to some embodiments of the present invention a method to aidin prognosis of a mammalian cancer patient is provided. This methodcomprises determining at least one of the following conditions: (a) in aspecimen of body fluid or secretion secreted by or bathing a mucosalepithelial tissue obtained from said patient, the lack of a cell growthinhibitory amount of at least one immunoglobulin inhibitor of steroidhormone responsive cell growth, (b) in a specimen of neoplastic cellsfrom said tissue, the loss or diminution of a TGFβ receptor, (c) in aspecimen of neoplastic cells from said tissue, the loss of a TGFβreceptor gene, (d) in a specimen of neoplastic cells from said tissue,the loss of heterozygosity for said TGFβ receptor gene, (e) in aspecimen of neoplastic cells from said tissue, the loss or diminution ofa poly-Ig receptor, (f) in a specimen of neoplastic cells from saidtissue, the loss of a poly-Ig receptor gene, (g) in a specimen ofneoplastic cells from said tissue, the loss of heterozygosity for saidpoly-Ig receptor gene, (h) in a specimen of neoplastic cells from saidtissue, the loss or diminution of a Fcγ receptor, (i) in a specimen ofneoplastic cells from said tissue, loss of a Fcγ receptor gene, and (j)in a specimen of neoplastic cells from said tissue, loss ofheterozygosity for said Fcγ receptor gene. The loss or diminution ineach instance is measured by comparison to similar determinations innon-neoplastic cells from the patient, and/or to the patient's previoustest results, and/or by comparison to predetermined standard values. Thepresence of one or more of the conditions is suggestive or indicative ofat least some degree of reduced prognosis of the patient, and an absenceof one or more of said conditions being suggestive or indicative of atleast some degree of favorable prognosis.

In accordance with certain other embodiments of the present invention, amethod to aid in suppressing or inhibiting malignant transformation orprogression in a steroid hormone responsive mucosal epithelial cell isprovided. The method comprises ensuring expression of a TGFβ receptor inthe cell sufficient to mediate TGFβ inhibition of neoplastic cellgrowth, and also ensuring expression of a poly-Ig receptor and/or a Fcγreceptor. In preferred embodiments, the method ensures that poly-Igreceptor is expressed in the cell sufficient to mediate IgA and/or IgMinhibition of steroid hormone responsive growth of the cell in theabsence of an inhibition reversing amount of the steroid hormone orsteroid hormone mimicking substance; and the Fcγ receptor is expressedsufficiently to mediate IgG1 inhibition of steroid hormone responsivegrowth of the cell in the absence of an inhibition reversing amount ofthe steroid hormone or steroid hormone mimicking substance. Ifexpression of one of those native receptors is lacking, it may benecessary to introduce an exogenous receptor gene using known genetransfer techniques.

In accordance with certain other embodiments of the invention, a methodof inhibiting or arresting in vivo cancer cell growth is provided. Themethod comprises contacting a steroid hormone responsive mucosalepithelial tissue with a pharmaceutical composition comprising apharmacologically acceptable carrier and at least one immunoglobulininhibitor of steroid hormone responsive cell growth chosen from thegroup consisting of IgA, IgM and IgG1, preferably dimeric or polymericIgA, polymeric IgM and IgG1. In some embodiments, the treatment methodalso includes administering an immunoglobulin inhibitor-mimickingsubstance such as tamoxifen or a metabolite thereof.

Certain other embodiments of the present invention provide a method oftreating cancer of a glandular or tissue that secretes or is bathed byan immunoglobulin, the method comprising enhancing the amount of atleast one immunoglobulin inhibitor of steroid hormone responsive cancercell growth secreted by or contacting the tissue. The inhibitor ispreferably IgA, IgM and IgG1.

According to certain embodiments of the present invention, a method oftreating cancer of a steroid hormone responsive mucosal/epithelialtissue is provided. In some embodiments, the method comprises detectingin a population of cancer calls obtained from the tissue the presence ofa poly-Ig receptor or a portion thereof. In some embodiments, the methodalso includes detecting in the population of cancer cells the presenceof ERγ. In still other embodiments, the method also includesadministering to an individual in need thereof, an effective amount ofan immunoglobulin mimicking substance (e.g., tamoxifen or a tamoxifenmetabolite) sufficient to inhibit cancer cell growth.

Although the cell growth inhibitory activity of the immunoglobulininhibitors is a function that is distinct from any additionalantibody-antigen recognition type immune activities, in some instancesconventional immunological techniques can be advantageously employed toproduce the desired inhibitors. Accordingly, in certain embodiments ofthe invention a method of inhibiting or arresting growth of a steroidhormone responsive tumor in a mammal is provided which includesadministering an immunogen to the mammal in an amount sufficient toinduce plasma and/or mucosal production of at least one secretoryimmunoglobulin inhibitor of steroid hormone responsive cell growthsufficient to inhibit steroid hormone responsive proliferation of aplurality of steroid hormone responsive cancer cells in the mammal. Insome embodiments the mode of administration is oral. In certainembodiments, the method also includes determining an age range of themammal during which the native production of the inhibitor(s) in themammal is less than a predetermined value. An age range in which thereare low concentrations of natural immunoglobulin inhibitors may presenta window of increased susceptibility to mutagenic or other carcinogenicevents. Some embodiments provide for administering the immunogen at apredetermined time such that production of the inhibitor(s) by themammal during that window of susceptibility is enhanced.

In certain other embodiments of the invention a method of inducingnatural mucosal production of cancer deterring factors is provided. Themethod comprises parenteral administration to a mammal of an amount ofsecretory immunoglobulin-stimulating antigen sufficient to induce plasmaand/or mucosal production of a predetermined steroid hormone responsivecancer cell growth inhibiting amount of at least one secretoryimmunoglobulin IgA, IgM and IgG1.

In still other embodiments of the invention a method of enhancing levelsof cancer deterring factors in a body fluid bathing a gland or mucosaltissue is provided. The method includes introducing into the body of anindividual in need thereof at least one exogenous steroid hormoneresponsive cell growth immunoglobulin inhibitor. In preferredembodiments the inhibitor(s) is/are IgA, IgM and IgG1. In someembodiments, the method also includes qualitatively and/orquantitatively testing a body fluid or secretion, such as saliva, forsaid at least one inhibitor to confirm immunization.

In accordance with still other embodiments of the invention, a method ofrestoring or enhancing immunoglobulin regulation of steroid hormoneresponsive cell growth in a mucosal epithelial cell is provided. Themethod comprises restoring or enhancing expression in the cell of amediator of immunoglobulin regulation chosen from the group consistingof a poly-Ig receptor and a Fcγ receptor. In some embodiments the methodcomprises inserting a gene for a poly-Ig receptor into said cell andexpressing said gene.

Also provided in accordance with certain embodiments of the presentinvention is a method of identifying carcinogenic bacteria. In certainembodiments the method includes: (a) obtaining a bacteria-containingspecimen of glandular/mucosal epithelial tissue or body fluid secretedby or bathing a gland or mucosal epithelial tissue; (b) takingprecautions in obtaining and handling said specimen such thatcontamination by extraneous microorganisms is avoided; (c) culturing thebacteria in said specimen such that at least one isolated bacterialcolony is obtained; (d) selecting at least one of said bacterialcolonies for further examination; and (e) conducting an Ames Test oneach selected colony such that mutagen-producing bacterial isolates areidentifiable. In certain embodiments the method also contains one ormore of the following steps: (f) determining the gram stain negative orgram stain positive classification of said bacterial colonies; (g)testing the bacterial isolates for production of defined metabolitesknown to or suspected of being mutagenic; (h) testing the bacterialisolates for induction of an oxidative burst when incubated with aneutrophil or macrophage; and (i) testing the bacterial isolates forimmunoglobulin protease activity. In some embodiments the method alsocontains one or more of the following steps: (j) when the fluidcomprises a breast secretion, determining whether a bacterial isolatesurvives and grows in the presence of a normal bacterial cell inhibitingamount of lactoferrin; (k) growing a bacterial isolate in a medium and,after growing the bacterial isolate, testing the medium with anon-tumorigenic human mucosal epithelial cell line such that cells thatare altered to a malignant phenotype by a component of the medium aredetectable; and (l) identifying a bacterial isolate using a polymerasechain reaction (PCR) technique.

In accordance with certain additional embodiments of the invention, amethod of conferring or enhancing resistance by a mucosal epithelialcell to malignant transformation is provided. The method comprisesinducing immunity in a host to at least one bacteria known to orsuspected of being oncogenic, as identified by the above-describedmethod.

In some embodiments of the invention a method of deterring malignanttransformation of a mucosal epithelial cell is provided that includesadministering an effective amount of an antibiotic to a host infected byan oncogenic bacteria, as identified by the above-described method.

In certain other embodiments, a method of suppressing an effect ofmalignant transformation of a mucosal epithelial cell is provided inwhich a cell growth arresting amount of at least one immunoglobulinchosen from the group consisting of dimeric or polymeric IgA, polymericIgM and IgG1 is administered to an individual in need thereof.

Still other embodiments of the present invention provide a method ofpreparing an anti-cancer antibody comprising selecting at least onebacteria known to, or suspected of, inducing malignant transformation inmucosal epithelial cells, according to the above-described method, andinducing immunity to the bacteria in an individual considered to be atrisk of developing cancer in a tissue comprising the cells.

Also provided in accordance with certain embodiments of the invention isa method of preventing or reducing the risk of developing cancer in amucosal epithelial tissue comprising immunizing an individual against atleast one bacteria known to or suspected of inducing malignanttransformation in that tissue. Preferably the bacteria is identified aspreviously described. In certain embodiments, the immunization comprisesorally, nasally or rectally administering an inactivated or attenuatedform of the bacteria to the individual such that mucosal immunityagainst the bacteria is conferred.

In certain embodiments of the invention, a method of suppressing aneffect of malignant transformation of a steroid hormone responsiveepithelial cell is provided. Representative steroid hormone responsiveepithelial cells are breast, prostate, oral cavity mucosa,salivary/parotid glands, esophagus, stomach, small intestine, colon,tear ducts, nasal passages, liver and bile ducts, bladder,secretory/exocrine pancreas, adrenals, kidney tubules, glomeruli, lungs,ovaries, fallopian tube, uterus, cervix, vagina, and secretory anteriorpituitary gland cells. The method comprises enhancing the amount of IgAand/or IgM and/or IgG1 secreted by or contacting the cell such thatsteroid responsive growth stimulation of the cell is inhibited in theabsence of a inhibition reversing amount of the steroid hormone or asteroid hormone mimicking substance.

In accordance with certain additional embodiments of the presentinvention, a method of detecting previous or active infection by abacteria known to or suspected of being oncogenic in mucosal epithelialtissue is provided which includes detecting in plasma or a body fluid orsecretion an antibody against the bacteria. In preferred embodiments thebacteria known to or suspected of being oncogenic is identified inaccordance with the above-described screening method.

In certain embodiments of the present invention, a method of preventingor reducing the risk of occurrence of cancer of a mucosal epithelialtissue, such as breast, prostate, colon, kidney and ovary, is provided.The method includes administering to a mammalian subject in need thereofat least one of the following treatments: (a) administering anantibiotic active against at least one bacteria known to or suspected ofinducing malignant transformation in mucosal epithelial cells; and (b)administering an immunogen to said subject in an amount sufficient toinduce plasma and/or mucosal production of at least one secretoryimmunoglobulin inhibitor of steroid hormone responsive cell growthsufficient to inhibit steroid hormone responsive proliferation of aplurality of steroid hormone responsive cancer cells in said mammal;administering at least one immunoglobulin inhibitor of steroid hormoneresponsive cell growth in an amount sufficient to inhibit or arreststeroid hormone responsive growth of said cells.

In accordance with certain other embodiments of the present invention, apharmaceutical composition is provided that comprises at least oneimmunoglobulin inhibitor of steroid hormone responsive cell growth and apharmacologically acceptable carrier. The cell is preferably a cancerousmucosal epithelial cell. In certain embodiments at least oneimmunoglobulin inhibitor is IgA, IgM or IgG1, preferably dimeric IgA,polymeric IgA, polymeric IgM or IgG1κ. In some embodiments thecomposition also contains one or more immunoglobulin inhibitor-mimickingsubstances, such as tamoxifen or a metabolite of tamoxifen.

Also provided in accordance with certain embodiments of the presentinvention is an anti-cancer composition comprising a pharmacologicallyacceptable carrier and a cytotoxic agent or a chemotherapeutic agentconjugated to an immunoglobulin inhibitor of steroid hormone responsivecancer cell growth. In preferred embodiments the inhibitor is IgA, IgM,IgG1, or any combination of those.

In accordance with certain other embodiments of the present invention amediator of steroid hormone reversible IgA and/or IgM inhibition ofsteroid hormone responsive cell growth is provided that comprises apoly-Ig receptor. In certain embodiments a mediator of steroid hormonereversible IgG1 inhibition of steroid hormone responsive cell growth isprovided which comprises a Fcγ receptor.

Also provided in certain embodiments of the present invention is anexpression vector for gene replacement therapy in a mammalian cell torestore or enhance expression of a poly-IgR. In some embodiments thevector comprises a preselected deoxyribonucleic acid (DNA) sequenceencoding a poly-Ig receptor, or a biologically active subunit or variantthereof, operably linked to a promoter capable of functioning in apreselected mammalian target cell. In some embodiments, an expressionvector for gene replacement therapy in a mammalian cell to restore orenhance expression of a Fcγ receptor is provided which comprises apreselected DNA sequence encoding a Fcγ receptor, or a biologicallyactive subunit or variant thereof, which is operably linked to apromoter functional in a preselected mammalian target cell.

Still other embodiments of the present invention provide an expressionvector for gene replacement therapy in a mammalian cell to restore orenhance expression of a TGFβ receptor. This vector comprises apreselected DNA sequence encoding a TGFβ receptor, or a biologicallyactive subunit or variant thereof, which is operably linked to apromoter functional in a preselected mammalian target cell.

Also provided in accordance with certain embodiments of the presentinvention is a method of expressing a DNA sequence encoding a mediatorof immunoglobulin inhibition of cell growth, the mediator being chosenfrom among a poly-Ig receptor, a Fcγ receptor, and biologically activesubunits and variants thereof operably linked to a promoter that iscapable of functioning in a preselected mammalian target cell. Themethod includes introducing the DNA sequence and the linked promoterinto the mammalian cell and allowing the cell to express the DNAsequence. In certain embodiments the also includes expressing a DNAsequence encoding a TGFβ receptor, or a biologically active subunit orvariant thereof, which is operably linked to a promoter that is capableof functioning in a preselected mammalian target cell. In thisembodiment the TGFβ receptor DNA sequence and its linked promoter areintroduced into the mammalian cell and the cell is allowed to expressthe DNA sequence.

These and other embodiments, features and advantages of the presentinvention will become apparent with reference to the followingdescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

For the detailed descriptions of the preferred embodiments, referencewill now be made to the accompanying figures which include graphs,charts, and test results:

FIG. 1. CDE-horse Serum Effect on MTW9/PL2 Cell Growth±10 nM E₂ for 7days. (A) Dose-response data expressed as cell numbers; (B)Dose-response data expressed as cell population doublings (CPD) per 7days.

FIG. 2. Restoration of Growth by Addition of 10 nM E₂ on days 0, 2, 4and 6 After Seeding the MTW9/PL2 cells into Inhibitory Medium Containing50% (v/v) of CDE-horse serum.

FIG. 3. Dose-Response Effects of Steroid Hormones on Growth of theMTW9/PL2 Cells in Medium Containing 50% (v/v) CDE-horse Serum.

FIG. 4. MTW9/PL2 Cell Growth±E₂ in Medium with CDE Sera from SeveralSpecies. (A) CDE-porcine Serum; (B) CDE-pregnant Human Serum; (C)CDE-adult Rat Serum; (D) CDE-adult Bovine Serum; (E) CDE-fetal BovineSerum; (F) CDE-fetal Horse Serum.

FIG. 5. CDE-horse Serum Effect on GH₄C₁ Cell Growth±10 nM E₂ for 10days.

FIG. 6. CDE-horse Serum Effect on ZR-75-1 Cell Growth±10 nM E₂ for 14days.

FIG. 7. CDE-horse Serum Effect on MCF-7A Cell Growth±10 nM E₂ for 10days.

FIG. 8. Kinetics of T47D Cell Growth in CDE-horse Serum±10 nM E₂. (A)Growth Kinetics in 20% CDE-horse±E₂ versus 10% Fetal Bovine Serum; (B)Growth Kinetics in 50% CDE-horse Serum±E₂.

FIG. 9. Rodent and Human ER⁺ Cell Line Growth in 50% CDE-human Serum±E₂.(A) T47D Human Breast Cancer Cells; (B) LNCaP Human Prostate CancerCells; (C) MTW9/PL2 Rat Mammary Tumor Cells; (D) GH₃ Rat Pituitary TumorCells; (E) GH₄C₁ Rat Pituitary Tumor Cells (F) H301 Syrian HamsterKidney Tumor Cells.

FIG. 10. Dose-Response of Steroid Hormones with T47D Cells in 50%CDE-horse Serum.

FIG. 11. Dose-Response of Steroid Hormones with GH₄C₁ Cells in 50%CDE-horse Serum.

FIG. 12. Dose-Response of Steroid Hormones with H301 Cells in 50%CDE-horse Serum.

FIG. 13. Dose-Response of Steroid Hormones with LNCaP Cells in 50%CDE-horse Serum.

FIG. 14. T₃ Growth Effects with GH₃ Cells in Serum-free Medium (PCM).

FIG. 15. E₂ Growth Effects with GH₃ Cells in Serum-free Medium (PCM)Minus E₂.

FIG. 16. T₃ Growth Effects with Three GH Cell Lines in 2.5% CDE-horseSerum.

FIG. 17. T₃ Growth Effects with Two GH Cell Lines in 50% CDE-horseSerum.

FIG. 18. Effect of XAD-4™ Resin Treated Horse Serum on MTW9/PL2 CellGrowth±E₂.

FIG. 19. Effect of XAD-4™ Resin Treated Horse Serum on T47D CellGrowth±E₂.

FIG. 20. Effect of Phenol Red on Estrogen Responsive MCF-7 Cell Growth.(A) MCF-7A Cell Growth in CDE-horse Serum±Phenol Red and ±E₂; (B)Estrogenic Effects with MCF-7A Cells±Phenol Red; (C) MCF-7K Cell Growthin CDE-horse Serum±Phenol Red and ±E₂; (D) Estrogenic Effects withMCF-7K Cells±Phenol Red.

FIG. 21. Effect of Phenol Red on Estrogen Responsive T47D and ZR-75-1Cell Growth; (A) T47D Cell Growth in CDE-horse Serum±Phenol Red and ±E₂;(B) Estrogenic Effects with T47D Cells±Phenol Red; (C) ZR-75-1 CellGrowth in CDE-horse Serum±Phenol Red and ±E₂; (D) Estrogenic Effectswith ZR-75-1 Cells±Phenol Red.

FIG. 22. Effect of Phenol Red on Estrogen Responsive MTW9/PL2 CellGrowth; (A) MTW9/PL2 Cell Growth in CDE-horse Serum±Phenol Red and ±E₂;(B) Estrogenic Effects with MTW9/PL2 Cells±Phenol Red.

FIG. 23. Dose-Response Effects of Phenol Red versus E₂ with Three ER⁺Cell Lines. (A) Growth Effects of Phenol Red with MCF-7K, T47D andMTW9/PL2 Cells; (B) Growth Effects of E₂ with MCF-7K, T47D and MTW9/PL2Cells.

FIG. 24. Estrogen Induction of Progesterone Receptors by Phenol Redversus E₂. (A) Induction by E₂ with T47D Cells; (B) Induction by PhenolRed with T47D Cells.

FIG. 25. Effects of TGFβ1 on Cell Growth in 2.5% CDE-horse Serum±E₂. (A)MCF-7K Cell Growth; (B) MTW9/PL2 Cell Growth.

FIG. 26. TGFβ1 Inhibition of ER⁺Rodent and Human Cell Line Growth±E₂.(A) Inhibition Data±E₂ Presented in Cell Number; (B) Inhibition Data±E₂Presented in CPD.

FIG. 27. EGF and TGFα as Substitutes for the Effects of E₂ in CDE-horseSerum. (A) MCF-7A Cell Growth; (B) MCF-7K Cell Growth; (C) T47D CellGrowth; (D) ZR-75-1 Cell Growth.

FIG. 28. IGF-I as a Substitute for the Effects of E₂ in CDE-horse Serum.(A) MCF-7K Cell Growth; (B) MCF-7A Cell Growth; (C) T47D Cell Growth.

FIG. 29. Growth of T47D Human Breast Cancer Cells in Standard and“low-Fe” D-MEM/F-12.

FIG. 30. Growth of LNCaP Human Prostate Cancer Cells in Standard and“low-Fe” D-MEM/F-12.

FIG. 31. Growth of MDCK Dog Kidney Tubule Cells in Standard and “low-Fe”D-MEM/F-12.

FIG. 32. Growth of AR⁺ LNCaP Cells in CAPM±DHT versus Growth inD-MEM/F-12 Containing 10% Fetal Bovine Serum.

FIG. 33 Growth of the AR⁻ DU145 and AR⁻ PC3 Cells in CAPM versus Growthin D-MEM/F-12 Containing 10% Fetal Bovine Serum.

FIG. 34. Dose-Response Effects of Individual Components of CAPMSerum-free Defined Medium on LNCaP Cell Growth.

FIG. 35. Effects of Deletion of Individual Components from CAPMSerum-free Medium on LNCaP, DU145 and PC3 Cell Growth±DHT.

FIG. 36. Effect of Fe (III) on MCF-7A Cell Growth in DDM-2MF Serum-freeDefined Medium.

FIG. 37. Effect of Fe (III) on T47D Cell Growth in DDM-2MF Serum-freeDefined Medium.

FIG. 38. Effect of Fe (III) on LNCaP Cell Growth in CAPM PlusApotransferrin.

FIG. 39. Comparative Effect of Fe (III) on LNCaP, DU145 and PC3 CellGrowth in CAPM.

FIG. 40. Growth Restoring Effect of Fe (III) Chelators in serum-freemedium with T47D Cells.

FIG. 41. Growth Restoring Effect of Fe (III) Chelators in serum-freemedium with LNCaP Cells.

FIG. 42. Comparison of DU145 Cell Growth in “low-Fe” and “standard”D-MEM/F-12 Based Serum-free Defined Medium CAPM.

FIG. 43. Comparison of PC3 Cell Growth in “low-Fe” and “standard”D-MEM/F-12 Based Serum-free Defined Medium CAPM.

FIG. 44. Growth of the DU145 Cells in CDE-horse Serum±DHT.

FIG. 45. Growth of the PC3 Cells in CDE-horse Serum±DHT.

FIG. 46. Growth of the ALVA-41 Cells in CDE-horse Serum±DHT.

FIG. 47. Comparison of Estrogenic Effects in Serum-free Defined Mediumand in D-MEM/F-12 Medium Supplemented with CDE-Horse Serum; (A) MCF-7KCell Growth in Serum-free Defined Medium±E₂; (B) MCF-7K Cell Growth inD-MEM/F-12 with CDE-horse Serum±E₂; (C) T47D Cell Growth in Serum-freeDefined Medium±E₂; (D) T47D Cell Growth in D-MEM/F-12 with CDE-horseSerum±E₂; (E) LNCaP Cell Growth in Serum-free Defined Medium±E₂; (F)LNCaP Cell Growth in D-MEM/F-12 with CDE-horse Serum±E₂.

FIG. 48. Effect of CDE-horse Serum on LNCaP Cell Growth in Serum-freeCAPM±E₂ and ±DHT.

FIG. 49. Comparison of Estrogenic Effects in Serum-free Defined Medium.

and in D-MEM/F-12 Medium Supplemented with CDE-Horse Serum.

GH₄C₁ Cell Growth in Serum-free Defined Medium±E₂.

GH₄C₁ Cell Growth in D-MEM/F-12 with CDE-horse Serum±E₂.

MTW9/PL2 Cell Growth in Serum-free Defined Medium±E₂.

MTW9/PL2 Cell Growth in D-MEM/F-12 with CDE-horse Serum±E₂.

H301 Cell Growth in Serum-free Defined Medium±E₂.

H301 Cell Growth in D-MEM/F-12 with CDE-horse Serum±E₂.

FIG. 50. Comparison of the Inhibitor Reversing Effects of DHT, E₂, andDES on LNCaP. Cell Growth in CDE-horse Serum Containing Medium; (A)Effect of DHT as an Inhibitor Reversing Steroid; (B) Effect of E₂ as anInhibitor Reversing Steroid; (C) Effect of DES as an Inhibitor ReversingSteroid; (D) Effect of Combinations of DHT, E₂, and DES as InhibitorReversing Steroids.

FIG. 51. Column Elution Profiles of the Two-step Cortisol Affinity andphenyl Sepharose Elution of CA-PA-pool I and CA-PS-pool II.

FIG. 52. Identification of the Molecular Forms Present in ActiveCA-PS-pool II. (A) SDS-PAGE with Coomassie Blue Staining; (B) WesternAnalysis with Anti-human SHBG.

FIG. 53. CA-PS-pool II Effect on ER⁺ Cell Growth in 2.5% CDE-horseSerum±E₂. (A) GH₁ Cells; (B) GH₃ Cells; (C) GH₄C₁ Cells; (D) H301 Cells;(E) MTW9/PL2 Cells; (F) MCF-7K Cells; (G) ZR-75-1 Cells (II) T47D Cells.

FIG. 54. Cortisol Affinity Column Depletion of the Estrogenic Activityin CDE-horse Serum Assayed with ER⁺ Cell Lines±E₂. (A) T47D CellsPre-Column; (B) T47D Cells Post-Column; (C) GH₃ Cells Pre-Column; (D)GH₃ Cells Pre-Column; (E) H301 Cells Pre-Column; (F) H301 CellsPost-Column.

FIG. 55. Serum-free Growth of Cells in Four Different Defined Media±E₂.(A) MTW9/PL2 Cells in DDM-2A; (B) T47D Cells in DDM-2MF; (C) GH₄C₁ Cellsin PCM-9; and (D) H301 Cells in CAPM.

FIG. 56. Effects of CDE-horse Serum on Estrogen Responsiveness of ThreeER⁺ Cell Lines Growing in Serum-free Defined Media. (A) T47D Cells inDDM-2MF; (B) MTW9/PL2 Cells in DDM-2A; (C) GH₄C₁ Cells in PCM-9.

FIG. 57. Effects of CA-PS-pool II on the Growth of Eight ER⁺ Cell Linesin Serum-free Defined Medium±E₂.

FIG. 58. Western Analysis of CA-PS-pool I and CA-PS-pool II with theAntibody Raised to the 54 kDa Band.

FIG. 59. Effect of the Anit-54 kDa Antiserum on the Inhibition ofMWT9/PL2 Cell Growth by the Isolated Fraction CS-PS-Pool II

FIG. 60. Western Immunoblotting of Commercially Prepared Horse IgG, IgAand IgM with anti-54 kDa Antiserum.

FIG. 61. Effect of Horse IgG on MTW9/PL2 Cell Growth in 2.5% CDE-horseSerum±E₂.

FIG. 62. Effect of Horse IgM on MTW9/PL2 Cell Growth in 2.5% CDE-horseSerum±E₂.

FIG. 63. Effect of Horse IgA on MTW9/PL2 Cell Growth in 2.5% CDE-horseSerum±E₂.

FIG. 64. SDS-PAGE with Coomassie Staining and Western Analysis of RatPurified “SHBG-like” Proteins. (A) SDS-PAGE of Purified RatPreparations; (B) Western Analysis with Anti-rat IgG.

FIG. 65. Western Analysis of a Rat Purified “SHBG-like” Preparation. (A)Western with Anti-rat IgA with Purified IgA Control; (B) Western withAnti-rat IgG1 with Purified IgG1 Control; (C) Western with Anti-rat IgMwith Purified IgM Control.

FIG. 66. Comparison of Rat IgG Subclasses for Antibody Cross-Reaction.(A) SDS-PAGE with Coomassie Blue Staining; (B) Western Analysis withRabbit Anti-Human SHBG.

FIG. 67. Effect of Rat IgG on MTW9/PL2 Cell Growth in Medium with 2.5%CDE-rat Serum±E₂.

FIG. 68. Effect of Rat IgA on MTW9/PL2 Cell Growth in Medium with 2.5%CDE-rat Serum±E₂.

FIG. 69. Effect of Rat IgM on MTW9/PL2 Cell Growth in Medium with 2.5%CDE-rat Serum±E₂.

FIG. 70. Mannan Binding Protein Isolation of Human Plasma/Serum IgM.

FIG. 71. Jacalin Lectin Purification of Human Plasma/Serum IgA.

FIG. 72. Effect of Human IgM on MTW9/PL2 Cell Growth±E₂ in Serum-freeDefined Medium.

FIG. 73. Comparison of the Effects of Rat and Horse IgA and IgM onMTW9/PL2 Cell Growth±E₂ in Serum-free Defined Medium Expressed in CellNumber and CPD.

FIG. 74. Effect of Rat Myeloma IgA on GH₁ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 75. Effect of Human Plasma IgA on GH₁ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 76. Effect of Human Plasma IgM on GH₁ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 77. Effects of sIgA on GH₁ Cell Growth in Serum-free DefinedMedium±E₂.

FIG. 78. Model of Mucosal Epithelial Cell Transport of IgA/IgM.

FIG. 79. Essential Structures of Human Plasma and Secretory IgA.

FIG. 80. Effect of Rat Myeloma IgA on GH₃ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 81. Effect of Rat IgM on GH₃ Cell Growth in Serum-free DefinedMedium±E₂.

FIG. 82. Effect of Human Plasma IgA on GH₃ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 83. Effect of Human Plasma IgM on GH₃ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 84. Effect of Human Secretory IgA on GH₃ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 85. Effect of Rat Myeloma IgA on GH₄C₁Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 86. Effect of Rat Plasma IgM on GH₄C₁Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 87. Effect of Human Plasma IgA on GH₄C₁Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 88. Effect of Human Plasma IgM on GH₄C₁Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 89. Effect of Human Secretory IgA on GH₄C₁Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 90. Effect of Mouse IgA on H301 Cell Growth in Serum-free DefinedMedium±E2.

FIG. 91. Effect of Human IgA on H301 Cell Growth in Serum-free DefinedMedium±E2. (A) Plasma IgA Effects; (B) Secretory sIgA Effects.

FIG. 92. Dose-Response Effects of E₂ on H301 Cell Growth in Serum-freeDefined Medium Containing 40 μg/mL Human Plasma IgM.

FIG. 93. Effect of Human IgA on MCF-7A Cell Growth in Serum-free DefinedMedium±E2. (A) Plasma IgA Effects; (B) Secretory sIgA Effects.

FIG. 94. Effect of Human IgA on MCF-7K Cell Growth in Serum-free DefinedMedium±E2. (A) Plasma IgA Effects; (B) Secretory sIgA Effects.

FIG. 95. Effect of Human IgM on MCF-7A Cell Growth in Serum-free DefinedMedium±E₂.

FIG. 96. Effect of Human IgM on MCF-7K Cell Growth in Serum-free DefinedMedium±E₂.

FIG. 97. Dose-Response Effects of E₂ on MCF-7K Cell Growth in Serum-freeDefined Medium Containing 40 μg/mL Human Plasma Ig.

FIG. 98. Effect of Human IgA on T47D Cell Growth in Serum-free DefinedMedium±E2. (A) Plasma IgA Effects; (B) Secretory sIgA Effects.

FIG. 99. Effect of Human IgM on T47D Cell Growth in Serum-free DefinedMedium E₂.

FIG. 100. Dose-Response Effects of E₂ on T47D Cell Growth in Serum-freeDefined Medium Containing 40 μg/mL Human Plasma IgM.

FIG. 101. Effect of Human. IgA on ZR-75-1 Cell Growth in Serum-freeDefined Medium±E2. (A) Plasma IgA Effects; (B) Secretory sIgA Effects.

FIG. 102. Effect of Human IgM on ZR-75-1 Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 103. Effect of Human IgM on HT-29 Cell Growth in Serum-free DefinedMedium±T3.

FIG. 104. Effect of Human IgA on LNCaP Cell Growth in Serum-free DefinedMedium±E2. (A) Plasma IgA Effects; (B) Secretory sIgA Effects.

FIG. 105. Effects of Human Plasma versus Human Myeloma IgM on LNCaP CellGrowth in Serum-free Defined Medium±DHT.

FIG. 106. Summary of Estrogenic Effects with Various ER⁺ Cell lines andDifferent Ig Sources.

FIG. 107. Effect of Tamoxifen on T47D Cell Growth in Serum-free DefinedMedium.

FIG. 108. Estrogen Reversal of Tamoxifen Inhibition of T47D cells inSerum-free Defined Medium.

FIG. 109. Estrogen Rescue of MTW9/PL2 Cell Growth in Serum-free MediumContaining 40 μg/mL of Horse Serum IgM.

FIG. 110. Summary of Estrogen Rescue of MTW9/PL2 Cell Growth inSerum-free Medium Containing 40 μg/mL of Horse Serum IgM.

FIG. 111. Estrogen Rescue of T47D Cell Growth in Serum-free MediumContaining 40 μg/mL of Human Serum IgM.

FIG. 112. Estrogen Rescue of MCF-7A Cell Growth in Serum-free MediumContaining 40 μg/mL of Human Serum IgM.

FIG. 113. Western Detection of the Secretory Component of Human MilksIgA.

FIG. 114. Effect of Anti-Secretory Component on IgM Inhibition of T47DCell Growth in Serum-free Defined Medium.

FIG. 115. Effect of Anti-Secretory Component on pIgA Inhibition of LNCaPCell Growth in Serum-free Defined Medium.

FIG. 116. Western Analysis with Anti-Secretory Component to Detect thePoly-Ig Receptor in AR⁺ and AR⁻ Prostate Cancer Cells plus Control CellLines.

FIG. 117. Effect of Human pIgA on DU145 Cell Growth in Serum-freeDefined Medium±DHT.

FIG. 118. Effect of Human pIgA on PC3 Cell Growth in Serum-free DefinedMedium±DHT.

FIG. 119. Effect of Rat Immunoglobulins on Estrogen Responsive Growth ofMTW9/PL2 Cells In Serum-free Defined Medium.

FIG. 120. Comparison of the Estrogenic Effects of Human Immungobulinwith T47D Cells in Serum-free Defined Medium.

FIG. 121. Effect of Human IgG Isotypes on LNCaP Cell Growth inSerum-free Defined Medium±DHT.

FIG. 122. IgG Isotype Assays with LNCaP Cells in Serum-free DefinedMedium±DHT.

FIG. 123. Model of Early Onset Breast Cancer Including TGFβ.

FIG. 124. Effect of Carcinogens on Mammary Tumor Induction in Rats ofVarious Ages.

FIG. 125. Anti-human SHBG Antibody Immunoprecipitation of the EstrogenicActivity Present in CDE-horse Serum Assayed with MTW9/PL2 Cells.

FIG. 126. Anti-human SHBG Antibody Immunoprecipitation of the EstrogenicActivity Present in CDE-rat Serum Assayed with MTW9/PL2 Cells.

FIG. 127. Anti-human SHBG Antibody Immunoprecipitation of the LabeledSteroid Hormone Binding Activity Present in CDE-rat Serum.

FIG. 128. Western Analysis and Densitometry of the Immunoglobulin Levelsin the Serum of Female Rats of Specified Age Groups.

FIG. 129. Structural and Functional Organization of the Human EstrogenReceptor α.

FIG. 130. Entre Genome NCBI Search of “Breast Cancer Mutations” andChromosomes.

FIG. 131. Chromosome 0.1 Map of Breast Cancer Loci versus the Poly-IgReceptor Locus.

FIG. 132. Colon, Breast and Prostate Cancer Death Rates Around theWorld.

FIG. 133. Immunoglobulin IgG, IgA and IgM Concentrations in Plasmaversus Human Age.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

To facilitate review of the detailed description of preferredembodiments, a Table of Contents is provided. The titles used for thevarious subsections and examples are not intended to be limiting and areonly an aid to locating certain subject matter. In addition, some of theExamples that follow begin with a short introduction and/or summary,which is intended merely to facilitate review and is not limiting on thedisclosure contained in the full Example, and end with a Discussion,which may include some conclusions that may be drawn from that Example.

Table of Contents Subsection Paragraph No. Introduction 199 Example 1:Methods and Compositions For Demonstration of Steroid 206 HormoneResponsive Cancer Cell Growth in Culture Example 2: Preparations ofSteroid Hormone Depleted Serum 224 Example 3: MTW9/PL2 Rat Mammary TumorCell Estrogen Responsive Growth in 34° C. 230 Charcoal-dextran ExtractedSerum Example 4: Estrogen Responsive Growth of Additional Rodent andHuman Cell Lines In 34° C. 245 Charcoal-dextran Extracted Horse andHuman Serum Example 5: Thyroid Hormone Growth Effects in CDE-Horse SerumPrepared at 34° C. 257 Example 6: Estrogenic Effects in XAD-4 ™ ResinTreated Horse Serum 259 Example 7: Testing of Substances for EstrogenicActivity 261 Example 8: Roles of TGFβ and Growth Factors: ConceptualImplications 275 Example 9: Serum-free Defined Culture MediumCompositions 293 Example 10: Serum-free Defined Medium Supports BothHormone Sensitive 316 and Autonomous Cancer Cells Example 11:Differential Effects of Fe (III) on the Growth of Hormone Responsive and322 Autonomous Human Breast and Human Prostate Cancer Cells Example 12:Growth in Serum-free Defined Medium versus Growth in CDE-Serum ± E₂ 332Example 13: Action of DES on Human AR ⁺LNCaP Prostate Cancer Cells 341Example 14: Properties and Rationale For Serum Purification Source 343Example 15: Cortisol Affinity and Phenyl Sepharose Isolation of the“SHBG-like” 347 Estrogen Reversible Inhibitor from CDE-Horse SerumExample 16: Serum-free Assay Systems for Measuring Large MagnitudeSteroid Hormone 366 Mitogenic Responses with the Two-Step PurifiedInhibitor Example 17: Chemical and Immunological Properties of thePartially Purified CA-PS-Pool II 370 Inhibitors and Identification asIgA and IgM Example 18: Regulation of Steroid Hormone-responsive andThyroid Hormone-responsive 391 Cancer Cell Growth in Serum-free DefinedMedium by Secretory and Plasma Forms of IgA and Plasma and Cell CultureDerived IgM Example 19: A New High Estrogen Affinity Growth RegulatingEstrogen Receptor (ERγ) 407 Example 20: Effect of Tamoxifen Antiestrogenin Serum-free Defined Medium 419 Example 21: Effect of Long-TermExposure of Breast Cancer Cells to IgM Under 428 Serum-free DefinedConditions Example 22: The Role of the Poly-Ig Receptor in HormoneResponsive and 433 Autonomous Breast and Prostate Cell Growth RegulationExample 23: IgG1 and IgG2 as Immunoglobulin Regulators of Estrogen and445 Androgen Responsive Cancer Cell Growth Example 24: Mediation ofIgG1κ Effects by a Fc-like Receptor 452 Example 25. ImmunoglobulinInhibitors as Tools for Identifying the Receptors that 455 Mediate theIgA/IgM/IgG Cell Growth Regulating Effects Example 26: Conceptual Modelfor Cascading Loss of Immunoglobulin Control in Progression 466 fromNormal Cells to Steroid Hormone Responsive and Autonomous CancersExample 27: Role of TGFβ in Breast Cancer Predicts the CellularProgression 475 in Early Onset Breast Cancer Example 28: Windows ofBreast Susceptibility to Carcinogenesis and Mutation 480 and the Levelsof Immunoglobulin Inhibitors Example 29: Risk Factors: IgA/IgM BasedTest to Detect Lowered Levels of Steroid 492 Hormone Reversible CellGrowth Inhibitors in Plasma or Body Secretions Example 30: Risk Factors:IgA Deficiencies and Malignancies 498 Example 31: Risk Factors:Autoimmunity Test for Anti-IgA and IgM In Plasma 500 Example 32:Diagnostic and Prognostic Tools: Estrogen Receptor γ (ERγ) 505 Example33: Diagnostic, Prognostic and Treatment Decision Tools: Poly-IgReceptor 511 (or Poly-Ig like Receptor) Example 34: Diagnostic Tools:Monoclonal Antibodies to the Poly-Ig Receptor 517 and Breast CancerImaging Example 35: Diagnostic, Prognostic and Treatment Decision Tools:Fc-like 520 Receptor for IgG1/IgG2 Example 36: Diagnostic, Prognosticand Treatment Decision Tools: TGFβ Receptors 524 Example 37: AtaxiaTelangiectasia as an Example of a Human Genetic Disorder 529 with HighRates of Breast Cancer Coupled with an IgA Deficiency Example 38:Diagnostic and Predictive: Poly-Ig Receptor Based Genetic Screening 531for Breast, Prostate and other Mucosal Cancer Susceptibility Example 39:Treatment: Breast Cancer Prevention with Applications to Prostate Cancer543 and other Mucosal Cancers Example 40: Treatment: Rat Model forTesting Oral Immunization Effects on 558 Mammary Gland CarcinogenesisExample 41: Treatment: Bacterial Oncogenesis and Prevention by OralImmunization 573 Example 42: Treatment: Treatment of Steroid HormoneResponsive Breast or Prostate Cancer 601 by Administration ofIgA/IgM/IgG1 Example 43: Treatment: Monoclonal Antibodies that Mimic orblock IgA or IgM Binding 609 to the Poly-Ig Receptor Example 44:Treatment: Delivery of Chemotherapeutic Agents and Cytotoxins to CancerCells 616 via IgA/IgM/IgG1 or Monoclonal Antibodies to Poly-Ig Receptor

Introduction

In the course of searching for what causes the growth of estrogenresponsive breast and androgen responsive prostate cancers, it wasdiscovered that the secretory immune system plays a major role in thosediseases. More specifically, it was discovered that the secretory immunesystem (i.e., the immunoglobulins IgA, IgM and IgG1) provide negative(inhibitory) regulation of steroid hormone responsive mucosal epithelialcancer cell growth in serum-free model cell culture systems, includingbreast, prostate, pituitary, kidney, colon, and other glandular cancercells. Prior to that discovery, which is described in co-ownedconcurrently-filed U.S. Pat. No. ______ (Atty. Dkt. No.1944-00201)/PCT/US2001/______ (Atty. Mt. No. 1944-00202) entitled“Compositions and Methods for Demonstrating Secretory Immune SystemRegulation of Steroid Hormone Responsive Cancer Cell Growth,” herebyincorporated herein by reference, no cell growth regulating role wasknown for the secretory immune system. The secretory immune systemproduces predominantly dimeric/polymeric IgA, secretory IgA (sIgA),polymeric IgM, and IgG1. The discovery of immunoglobulin inhibitors ofcell growth is a major breakthrough in the understanding of cancers ofbreast and prostate, as well as other glandular/mucosal tissues thatsecrete or are bathed by the secretory immunoglobulins.

For the first time, a direct link is established between the secretoryimmune system and the most prevalent types of cancer that occurthroughout the world. Binding of IgA and IgM to the polyimmunoglobulinreceptor (poly-Ig receptor) is an important step in carrying out theregulatory function of IgA and IgM, and it is probable that the knownpoly-Ig receptor, or a closely related poly-Ig like receptor, mediatesthe negative regulation of steroid hormone dependent cell growth.Similarly, it is believed that the binding of IgG1 to the Fcλ receptoris a mediating step in carrying out the regulatory function of IgG1. Theapplication of this new understanding of immune system regulation ofcancer cell growth to the risk assessment, detection, diagnosis,prognosis, treatment and deterrence or prevention of a host of mucosalepithelial cell cancers is described in the following examples.

A new conceptual model described herein, offers an explanation of hownormal breast tissue may give rise to highly malignant, and dangerous,hormone autonomous forms. This model is contrary in some respects to thewell-established “linear progression” model, in which breast cancerspass through a characteristic natural history that involves a gradualevolution from near normal growth patterns into cancers that arecompletely steroid hormone autonomous (i.e., they are no longerstimulated by steroid hormones), and describes cell growth regulatoryroles for TGFβ, IgA, IgM and IgG1

The secretory immune system was not previously known to have any cellgrowth regulatory role in breast or prostate cancer, or in other cancersof the mucus epithelial tissues. As set forth in various of thefollowing examples, new compositions and “immunotherapy” protocols basedon production or administration of IgA, IgM and IgG1, as well as newmethods of immune-related diagnosis and assessment of susceptibility areprovided. Also, effective new gene expression and gene transfectiontherapies by which malignant breast cells may be returned to naturalimmune control are described. Such strategies will be far less toxicthan those now employing chemotherapeutic agents. A significant featureof the discovery is that a new natural “immune” mechanism exists that isdistinct from the anti-tumor immunological approaches of the past, andwhich can be exploited to control breast cancer.

It should be readily appreciated that this discovery has implicationswell beyond cancers of the breast and prostate. The secretory immunesystem is an integral part of the physiology of all mucosal epithelialtissues. Most, if not all, mucosal tissues secrete IgA, IgM and IgG1directly into the lumen of biological passageways. This includessalivary glands, oral and nasal cavities, stomach, small and largebowel, lung passageways, the kidney tubule, liver and bile ducts,prostate, bladder, the anterior pituitary, and the secretory/exocrinepancreas. Secretory immune system control of cell proliferation is alsorelevant to cancers of the female reproductive tract. The entire femalereproductive tract including ovaries, uterus, cervix and vagina eithersecretes IgA and IgM or is a target for these immunoglobulins. In fact,malignancies of all secretory epithelial tissues represent 80% or moreof the cancers in human females.

The compositions and methods, and the biochemical, genetic andimmunological tools described herein, and those described in U.S. Pat.No. ______ (Atty. Dkt. No. 1944-00201)/PCT/US2001/______ (Atty. Dkt. No.1944-00202) entitled “Compositions and Methods for DemonstratingSecretory Immune System Regulation of Steroid Hormone Responsive CancerCell Growth” (hereby incorporated herein by reference), are employed inthe present investigations to further elucidate the cascade of cellularchanges that lead to malignancy in glandular/mucosal tissues and toprovide, among other things, ways of testing cancer cells for loss ofIgA/IgM/IgG1 regulation, ways to detect genetic changes in the poly-Igreceptor, biochemical and genetic screening procedures to identifyindividuals at high risk for developing breast or prostate cancer, andways of deterring or reducing the risk of development of such cancers.Additionally, in light of the discovery that the secretory immune systemimmunoglobulins IgA, IgM and IgG1 are potent inhibitors of steroidhormone responsive cancer cell growth, it is now proposed that thesteroid hormone responsive tissues in the body can be protected from thecancer causing actions of certain environmental carcinogens, especiallyduring age related “windows” of increased susceptibility, by enhancementof the IgA/IgM/IgG1 secreted by or coming in contact with those tissues.In this way, DNA synthesis dependent mutations can be prevented orsubstantially reduced in those tissues. Likewise, deleteriousdown-modulation or inactivation of critical gene expression (e.g., thepoly-Ig receptor) due to environmental carcinogens may also beremediable by restoration of IgA, IgM and/or IgG1 control of cellgrowth.

Also described in examples that follow is the use of cell growthinhibitory amounts iron, in the form of Fe to treat malignancies and/orsurgical sites. Still other Examples which follow describe screeningprocedures for detecting potentially cancer-inducing bacteria, and offerpreventative measures for decreasing the effects of bacterialcarcinogesis.

EXAMPLES Example 1 Methods and Compositions for Demonstration of SteroidHormone Dependent Cancer Cell Growth in Culture

In the following Examples, which describe representative, preferredembodiments of the present invention, the following general materialsand methods are employed, except as otherwise noted therein.

Cell Culture Medium. The water used to prepare culture media and allother solutions was purified first by reverse osmosis followed bypassage through a U.S. Filter Corporation system with a charcoal filterand two mixed bed ion exchangers. The effluent was distilled using aBellco glass apparatus with quartz heating elements. The distilled waterwas stored in airflow restricted glass containers. No metal fittings areallowed in contact with the final purified water. This necessaryprecaution minimizes recontamination with metal ions. Standard phenolred containing Ham's F12-Dulbecco's modified Eagle's medium(D-MEM/F-12), phenol red-free standard D-MEM/F-12 and a custom-prepared“low-Fe” D-MEM/F-12 medium were supplied by Gibco-BRL (Catalog No.11330-032) or Bio♦Whittacker (Catalog No. 12-719, liquid). The “low-Fe”medium was standard phenol red containing D-MEM/F-12 from which theusual additions of ferric nitrate and ferrous sulfate had been omitted(Eby J E et al. (1992) Anal Biochem 203, 317-325; Eby J E et al. (1993)J Cell Physiol 156, 588-600). This medium was a special formulationpurchased from Gibco-BRL as a powder and prepared in the highly purifiedwater before 0.2 μm pore filter membrane sterilization. A number ofother stock solutions are required for cell culture in either serumcontaining or serum-free defined medium. Descriptions of eachpreparation are provided along with specific instructions for their use.The solutions used were designed to minimize the exogenous content ofsteroid hormone and to minimize the Fe (III) content of the water. Stepsare taken for the exclusion of all extraneous sources of steroidhormones and Fe (III). Exclusion of Fe (III) is highly preferred, and inmost of the totally serum-free applications, it is considered essential.Wherever possible, disposable plastic ware or glassware is used tominimize potential contamination. It is important to note that excesssolutions are preferably discarded after use with each individual cellline to avoid cross-contamination of cell types (Nelson-Rees W A andFladermeyer R R (1977) Science (Wash DC) 195, 134-136).

General Cell Culture—Serum. Adult and fetal horse, adult pig, adultsheep and adult and fetal bovine serum were obtained from Gibco-BRL. Amixture of adult male and female rat serum was purchased from Pel-Freez,Rodgers, AR. Human serum was purchased from Bio♦Whittacker. Human plasmawas a pool of samples collected from pregnant females during routinevisits to a local clinic. All serum was stored frozen at −20° C. untilused. Repeated freeze-thaw of serum or plasma is avoided. Beforecharcoal extraction, the EDTA was removed by dialysis at 7° C. for 24hours against forty volumes of 0.05 M Tris-HCl, pH 7.4, containing 50 mMCaCl₂. Dialysis was done with Spectropor 1 membranes (Spectrum MedicalIndustries, molecular weight cut-off 6,000 to 8,000). The clottedmaterial was removed by centrifugation. This preparation is termedplasma-derived serum. The serum or plasma was not heat pre-treated, orheat inactivated prior to use in the methods described below.

General Cell Culture—Normal Saline. Sterile normal saline (0.15 M NaCl)was prepared in 10 mL aliquots and stored at room temperature. Unusedportions are discarded at the end of each experiment. A large supply issterilized by autoclaving and used to prepare the solutions describedbelow.

General Cell Culture—Trypsin/EDTA for Subculture. Sterile preparationswere purchased from Irvine Scientific (Catalog No. 9341) orBio♦Whittacker (Trypsin-Versene EDTA Mixture) (Catalog No. 17-161F).This preparation contained 0.5 g/L trypsin and 0.2 g/L EDTA in Hank'sbalanced salts solutions with 10 mg/L phenol red. This preparation doesnot contain Ca or Mg salts nor does it have NaHCO₃. To trypsinize cells,1.5 mL of this preparation was typically used. Aliquots (2 mL) werestored frozen until used and residual solution discarded at the end ofeach experiment or application to a cell line.

General Cell Culture—Soybean Trypsin Inhibitor (STI). STI was purchasedfrom Sigma (Catalog No. T9128, Type II-2). An amount of 1.0 mg of thispreparation will inactivate 1.0 mg of trypsin activity. The solution isprepared as 0.2% (w/v) in normal saline and sterilized using a 0.2 μmpore diameter filter membranes. Aliquots of 3.0 mL are stored at −20° C.until used. This preparation is used to stop the action of trypsinduring harvest of stock cultures for growth assays. STI ensures that alltrypsin used to harvest cells for growth assays is inactivated andtherefore will not damage the protein additions to serum-free definedmedium. Also, use of STI ensures that no extraneous steroid hormones areintroduced after harvest of cells from the stock culture dishes.

General Cell Culture—Crude Pancreatic Trypsin for Cell Counting. Thistrypsin preparation was used to harvest the cells for determining cellnumbers. The cells are typically grown in 35-mm diameter dishes. Thisenzyme was purchased from ICN Biochemicals as the 1-300 porcinepancreatic trypsin preparation (Catalog No. 103140). A stock solution istypically prepared by adding the contents of a preweighed bottle of 1×Dulbecco's modified PBS medium without calcium or magnesium to 800 mL ofwater. This solution dissolves very gradually with adjustment to pH 7.3using NaOH. After the solution was clear, 20 g of crude trypsin wasadded and this mixture stirred for 30 minutes at room temperature. Thesomewhat cloudy solution was diluted to 1000 mL with water and thisvolume was stored frozen in bulk overnight at −20° C. to induce coldrelated precipitation that typically occurs when this preparation wasfrozen and thawed. After thawing at 37° C. in a water bath, thepreparation was filtered through 0.45 μm pore membranes. Thispreparation was stored at −20° C. in useable portions.

General Cell Culture—EDTA for Cell Counting. The EDTA used is thedisodium and dihydrate salt (Sigma Catalog No. E1644). A 0.29 M solutionis prepared by adding 107.9 g to 800 mL of water with stirring andadjustment to pH 7.2 with NaOH. The volume is brought to one liter withwater and the solution stored at room temperature. Because this solutionis used only at the end of the experiments, it does not requiresterilization.

General Cell Culture. In Table 1 the cell lines used in the describedExamples are listed. The abbreviation “KCC” is the Karmanos CancerCenter, Cell Line Repository, Detroit, Mich. The abbreviation “ATCC” isthe American Type Culture Collection, Cell Line Repository, Manassas,Va. Professor Armen Tashjian's address is Harvard University, Boston,Mass. Dr. William Rosner's address is Columbia University, New York. Dr.Sirbasku's address is The University of Texas, Houston, Tex. Thesuperscript designations in Table 1 for each of the cell lines indicatereferences that verify that the estrogen and androgen responsive celllines used in this study are bona fide hormone responsive based on theirtumor forming characteristics in host animals. Those reports are cleardemonstrations of the reliability of the models used in the presentinvestigations to study sex hormone dependence in culture.

TABLE 1 Cell Lines Employed in the Examples. ER⁺indicates receptorcontaining/E₂ sensitive CELL LINES SOURCES REFERENCES/CELL LINE ORIGINMCF-7K¹ KCC Soule HD et al. (1973) J Natl Cancer Inst 51, 1409-1416 ER⁺human breast cancer MCF-7A¹ ATCC Soule HD et al. (1973) J Natl CancerInst 51, 1409-1416 ER⁺ human breast cancer T47D² ATCC Keydar I et al.(1979) Eur J Cancer 15, 659-670 ER⁺ human breast cancer ZR-75-1³ ATCCEngle LW et al. (1978) Cancer Res 38, 3352-3364. ER⁺ human breast cancerGH₄C₁ ⁴ Dr. A. Tashjian Tashjian AH Jr (1979) Methods Enzymol 58,527-535 ER⁺ rat pituitary tumor GH₃ ⁵ ATCC Tashjian AH Jr (1979) MethodsEnzymol 58, 527-535. ER⁺ rat pituitary tumor GH₁ ATCC Tashjian AH Jr(1979) Methods Enzymol 58, 527-535 ER⁺ rat pituitary tumor MTW9/PL2⁶ Dr.D. Sirbasku Danielpour D et al. (1988) In Vitro Cell Dev Biol 24, 42-52ER⁺ rat mammary tumor H301⁷ Dr. D. Sirbasku Sirbasku DA and Kirkland WL(1976) Endocrinology 98, 1260-1272 ER⁺ Syrian hamster kidney tumorLNCaP⁸ ATCC Horoszewicz JS et al. (1983) Cancer Res 43, 1809-1818 AR⁺human prostatic carcinoma Fibroblasts Dr. D. Sirbasku Primary culturesof human foreskin and rat ear cartilage; Eastment CT and Sirbasku DA(1980) In Vitro 16, 694-705 ALVA-41 Dr. W. Rosner Nakhla AM and Rosner W(1994) Steroids 59, 586-589 AR⁻ human prostate cancer; androgen growthinsensitive DU145 ATCC Stone KR et al. (1978) Int J Cancer 21, 274-281AR⁻ human prostate cancer; androgen growth insensitive PC3 ATCC KaighnME et al. (1979) Invest Urol 17, 16-23 AR⁻ human prostate cancer;androgen growth insensitive HT-29 ATCC Chen TR et al. (1987) CancerGenet Cytogenet 27, 125-134 Thyroid hormone responsive human coloncancer ER⁺ indicates estrogen receptor containing. AR⁺ indicatesandrogen receptor containing. Unless otherwise noted, these designationsindicate sex steroid hormone growth responsive in culture.

In vivo Tumor Forming Properties. The references below refer to thesuperscript designations in Table 1 for the cell lines. The referencesverify that the estrogen and androgen responsive cell lines used in thisdisclosure are hormone responsive based on their tumor formingcharacteristics in host animals. These reports are demonstration thereliability of the models used in this disclosure to study sex hormonedependence in culture.

-   ¹The use of two strains of MCF-7 cells has been described (Sirbasku    D A and Moreno-Cuevas (2000) In Vitro Cell Dev Biol 36, 428-446).    Clonal variations of this line are known (Seibert K et al. (1983)    Cancer Res 43, 2223-2239). Demonstration of estrogen responsive    MCF-7 tumor formation in vivo (Huseby R A et al. (1984) Cancer Res    44, 2654-2659; Soule H D and McGrath C M (1980) Cancer Lett 10,    177-189; Welsch C W et al. (1981) Cancer Lett 14, 309-316).-   ²Estrogen responsive T47D tumors in vivo (Leung C K H and Shiu R P    C (1981) Cancer Res 41, 546-551).-   ³Estrogen responsive ZR-75-1 tumors in vivo (Osborne C K et    al. (1985) Cancer Res 45, 584-589).-   ⁴Estrogen responsive GH₄C₁ tumors in vivo (Riss T L and Sirbasku D    A (1989) In Vitro Cell Dev Biol 25, 136-142).-   ⁵Estrogen responsive GH₃ tumors in vivo (Sorrentino J M et    al. (1976) J Natl Cancer Inst 56, 1149-1154).-   ⁶Estrogen responsive MTW9/PL2 tumors in vivo (Sirbasku D A (1978)    Cancer Res 38, 1154-1165; Danielpour D and Sirbasku D A (1984) In    Vitro 20, 975-980).-   ⁷Estrogen responsive H301 tumors in vivo (Sirbasku D A and Kirkland    W L (1976) Endocrinology 98, 1260-1272; Liehr J G et al. (1986) J    Steroid Biochem 24, 353-356).-   ⁸Androgen responsive LNCaP tumors in vivo (Sato N et al. (1997)    Cancer Res 57, 1584-1589; Gleave M et al (1991) Cancer Res 51,    3753-3761; Horoszewicz J S et al. (1983) Cancer Res 43, 1809-1818;    Pretlow T G et al. (1991) Cancer Res 51, 3814-3817; Passaniti A et    al. (1992) Int J Cancer 51, 318-324).

General Cell Culture—Cell Passage Method. All stock cultures were grownin medium containing phenol red. Stocks of the cells were maintained at37° C. in a humid atmosphere of 5% (v/v) CO₂ and 95% (v/v) air in 17 to20 mL of standard D-MEM/F-12 with 2.2 g per liter sodium bicarbonate, 15mM HEPES (pH 7.4), and serum. With all cell lines except the ratpituitary cells, the serum used for stock culture was 10% (v/v) fetalbovine serum (FBS). For the three rat pituitary tumor cell lines GH₄C₁,GH₁ and GH₃, the medium contained 12.5% (v/v) horse serum and 2.5% (v/v)FBS. To passage the cells, the medium was removed and the dishes washedwith 10 ml, of saline. Next, the cells were dissociated by incubation atroom temperature or at 37° C. for 3 to 10 minutes with 1.5 mL oftrypsin/EDTA. The action of the trypsin was stopped by addition of 8 mLof D-MEM/F-12 containing 10% (v/v) FBS or 8 mL of the horse serum orFBS. The cells were collected by centrifugation at 1000×g for 5 minutesand suspended in 10 mL of fresh serum containing medium. Aliquots werediluted into Isoton II (Coulter Diagnostics) and cell numbers determinedwith a Model ZBI or Z1 Coulter Particle Counter. The new dishes (100-mmdiameter with 15 to 20 mL of fresh medium) were seeded with 2.0×10⁵ to1.0×10⁶ cells on an alternating three-four day schedule or weekly asdictated by cell line growth rate. Cultures were used for growth assaysbetween three and six days after passage. Acidic (yellow mediumindicator color) cultures are not used for growth assays.

General Cell Culture—Media Types Used. The assays done in the presenceof serum were initially in “low-Fe” D-MEM/F-12 containing phenol red(Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36,410-427). The issue of the significance of the presence or absence ofphenol red, a potential estrogen (Berthois Y et al. (1986) Proc NatlAcad Sci USA 83, 2496-2500), has been dealt with in considerable detail(Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36,447-464). The Fe (III) content of this medium was ≦0.2 μM (Eby J E etal. (1992) Anal Biochem 203, 317-325). Fe (III) levels of ≧1.0 μMinterfere with thyroid hormone and estrogen responsive rat pituitarytumor cell growth in culture (Eby J E et al. (1992) Anal Biochem 203,317-325; Eby J E et al. (1993) J Cell Physiol 156, 588-600; Sato H etal. (1991) In Vitro Cell Dev Biol 27A, 599-602; Sato H et al (1992) MolCell Endocrinol 83, 239-251). Although Fe (III) might prevent estrogenresponsiveness from being identified in culture with MTW9/PL2 cells, asshown herein and reported (Sirbasku D A and Moreno-Cuevas J E (2000) InVitro Cell Dev Biol 36, 428-446; Moreno-Cuevas J E and Sirbasku D A(2000) In Vitro Cell Dev Biol 36, 447-464), this is not the case whenserum is present. Standard Fe (III)/Fe (II) containing D-MEM/F-12 was aseffective as the low-Fe medium. It is clear that the apotransferrin inthe serum effectively reduced the free Fe (III) in the medium to lessthan cytotoxic levels. As stated above, apotransferrin binds Fe (III)with very high affinity at pH 7.4 in plasma. The total concentration oftransferrin in serum is about 3 mg/mL. Usually, two-thirds of the totalis apotransferrin. This amount is more than adequate to chelate Fe (III)in culture medium (Eby J E et al. (1992) Anal Biochem 203, 317-325).However, in assays in serum-free defined medium, as described below, aFe (III) chelator (e.g. apotransferrin or DFX) is present in theserum-free defined medium at sufficient levels to neutralize the toxiciron.

General Cell Culture—Growth Assay Methods. Cell growth assays wereinitiated with stock cultures that were harvested by trypsin/EDTAtreatment as described above with one exception. It was highly preferredto stop the action of trypsin with 3 mL, of soybean trypsin inhibitor(0.5% w/v in saline) instead of medium containing serum. The use oftrypsin inhibitor reduced the possibility of contamination of thesubsequent assay media by serum-derived steroid hormones. Thedissociated cells were collected by centrifugation as described aboveand washed three times with 10 mL volumes of serum-free standardD-MEM/F-12. After each wash, care was taken to aspirate all medium fromthe cell pellet and the walls of the centrifuge tubes. This minimizedthe carryover of steroid hormones into the experimental test dishes. Bytaking steps to avoid carryover of serum, steroid hormones are preventedfrom being retained by the cells in culture. It is highly preferred towash the cells in this way before assaying to measure various steroidhormone effects in culture. It has been reported that steroid hormonesare retained long term by breast cancer cells in culture (Strobl J S andLippman M E (1979) Cancer Res 39, 3319-3327). The above-described washprocedure negates this problem. After the final wash, the cells weresuspended in 10 mL of serum-free D-MEM/F-12 and cell numbers determined.When cells were to be assayed in medium without phenol red discussedelsewhere herein and reported (Moreno-Cuevas J E and Sirbasku D A (2000)In Vitro Cell Dev Biol 36, 447-464), the cells were washed andresuspended in phenol red free D-MEM/F-12 purchased from Gibco-BRL. Thegrowth assays were initiated in 35-mm dishes containing a total of 2.0mL of medium and the final concentration of all components exceptsteroid hormones. The steroid hormone stocks were diluted to appropriateconcentrations in serum-free D-MEM/F-12 and 20 μL aliquots added to eachdish. For all growth assays, the medium was not changed after theinitial inoculation. Because several of the cell lines described inTable 2 grow in serum containing medium and serum-free defined medium asmixtures of suspension and attached cells, removal or changing of themedium during the course of the assays causes substantial cell losses.For all cell growth assays, the initial seed densities ranged from 5,000to 12,000 cells per 35-mm diameter dish.

General Cell Culture—Steroid Hormone Preparations. A number of hormonepreparations are used to supplement the cell cultures. Unlabeled steroidhormones were obtained from Sigma or Steraloids. Stock solutions wereprepared in sterile glass containers. The powder (non-sterile) steroidis added to the bottle along with 200 ml of 70% aqueous ethanol (readyas sterile). The steroids dissolve within an hour at room temperature,or when required were dissolved by gentle heating on a hot plate (handtemperature test-no boiling-no open flames). The stock solutions werestored at 4° C. and renewed at six-month intervals. It is not necessaryor desirable to filter sterilize these solutions because of steroidhormone loss on filter membranes. Stocks of 1.0 mM steroid hormones wereprepared. To prepare diluted stocks for direct use in culture, 10 μL of1.0 mM steroid hormone is diluted into 10 mL of D-MEM/F-12. This gives astock of 1.0 μM. It is used in the assay dishes or diluted further inD-MEM/F-12 as needed. The diluted steroids are discarded after each usebecause they bind to the plastic with storage. The formula weight (FIAT)of each of the common natural and synthetic hormones used is listedbelow in Table 2 along the abbreviation used for each and the amountsrequired to prepare 200 mL of stock.

TABLE 2 Preparation of Steroid Hormone Stocks for Cell Culture andHormone Binding Assays FORMULA STEROID HORMONES WEIGHT (FW)MILLIGRAMS/200 mL 17β-estradiol (E₂) 272.4 54.4 Estrone (E₁) 270.4 54.1Estriol (E₃) 288.4 57.7 Diethylstilbestrol (DES) 268.4 53.7 TamoxifenCitrate (TAM) 563.6 112.7 Progesterone (PROG) 314.5 62.9Hydrocortisone/Cortisol (C) 362.5 72.5 Dexamethasone (DEX) 392.5 78.5Testosterone (T) 288.4 57.7 Dihydrotestosterone (DHT) 290.4 58.1

General Cell Culture—Harvest and Counting Cells. At the termination ofthe experiments, each plate received 0.4 mL of crude pancreatic trypsindissolved in phosphate buffered saline was added along with 0.3 mL of0.29 M EDTA. After 4 to 40 minutes incubation at room temperature or at37° C., the action of the trypsin was stopped by addition of 0.6 mL ofhorse serum. The cell clumps were dissociated further by one passagethrough a 20½ or 23-gauge needle and syringe. This suspension was thendiluted to 10 mL with Isoton II and cell numbers determined with aCoulter Counter. The results are presented as the average of triplicatedishes for each test medium. To determine day zero cell numbers, atleast triplicate 1.0 mL aliquots of the inoculum were collected forcounting during the seeding of the test dishes. Coulter Counterstandardization and monitoring were performed by the manufacturer.

General Cell Culture—Quantification of Growth. The cell number resultsare converted to cell population doublings (CPD) by the followingcalculation:

${CPD} = \frac{\frac{{Log}_{10}\mspace{14mu} {Average}\mspace{14mu} {Cell}\mspace{14mu} {Number}\mspace{14mu} {on}\mspace{14mu} {Collection}\mspace{14mu} {Day}}{{Log}_{10}\mspace{14mu} {Average}\mspace{14mu} {Cell}\mspace{14mu} {Number}\mspace{14mu} {on}\mspace{14mu} {Day}\mspace{14mu} {Zero}}}{{Log}_{10}2}$

For the purposes of this Disclosure, the mitogenic response to sexsteroid hormones is designated the “steroidogenic effect.”—For example,the “estrogenic effect” is calculated as the difference between CPDmeasured in the presence of an estrogen minus CPD in the absence of thesteroid. These values equal cell number increases of 2^(CPD). The term“androgenic effect” has the same meaning except that it describes growthcaused by androgens such as DHT and T. CPD is used herein as a measureof growth because it is a direct calculation of the number of times acell population undergoes cell division. Furthermore, CPD use permits adirect measure of ED₅₀ and ED₁₀₀ Concentrations in different and inreplicate assays. The significance of differences between test dishesand controls was evaluated by the student's t test. Values of p<0.05were accepted as significant. Standard deviations (±SD) are includedwhen appropriate.

Discussion of Example 1. The cell culture methods outlined above arehighly preferred as procedures to obtain cells sufficiently washed ofsteroid hormone to measure low concentration effects in medium withhormone depleted serum prepared as described in the next Example. Theuse of STI to stop the action of the trypsin is highly preferred.Application of serum to stop the action of the trypsin causes asubstantial loss of hormone responsiveness.

Example 2 Methods of Preparing Steroid Hormone Depleted Serum

In this example, two methods for preparing steroid hormone depletedserum are described. The primary purpose was to prepare serum thatsupported large magnitude sex steroid growth effects in culture and toidentify the dose-response concentrations that cause the effects, asdemonstrated in Examples that follow. This meant preparing serum with ≦5μg/mL estrogen (and other steroid hormones). This concentrationcorresponds approximately to the lower limit of detection of steroids byradio immunoassay. The methods tested included (A) a two-stepcharcoal/dextran extraction of serum at 34° C., and (B) a one steptreatment with Amberlite™ XAD™-4 resin at 37° C.

A. Charcoal-Dextran Extraction at 34° C.

Preparation of the charcoal/dextran mixture. Activated charcoal,untreated powder (100 to 400 mesh), was obtained from Sigma (Catalog No.C5260). This preparation was done at room temperature. The powder (30 g)was suspended in 600 mL of water and stirred for 20-30 minutes at roomtemperature. The water used to wash and suspend the charcoal was apurified source made by reverse osmosis/ion exchange treatment/charcoalfiltration/0.2 μm pore diameter filtration/distillation into glass(only) containers. Next, 3.0 g of Dextran T70 (Pharmacia) was dissolvedin 300 mL of water, added to the charcoal suspension with stirring, andthe mixture stirred for 30-60 minutes at room temperature, preferably 60minutes. The mixture was then washed with about 6-8 liters of distilledwater in a sintered glass funnel (2000 mL, ASTM 40-60, C#36060). Thiswash removes impurities as well as fine particles of charcoal thatcannot be separated from serum by centrifugation. The charcoal-dextranretentate was suspended in a final volume of 300 mL of distilled waterto yield a suspension of 100 mg/mL charcoal and 10 mg/mL dextran.Preferably the mixture is stirred vigorously for about an hour, and thenstored at room temperature for no more than about 2-3 weeks prior touse.

Charcoal-dextran extraction at 34° C. of horse serum (CDE-horse serum).This serum in 500 mL sterile bottles was removed from the freezer (−17°C.) and thawed at 4° C. for 24 to 48 hours. Alternatively, fresh serumcould be used. The thawed serum (still in the 500 mL sterile bottles)was placed in an orbital shaker water bath (Lab-Line Orbit Shaker WaterBath) equilibrated at 34° C. The serum was incubated at 140 RPM for45-60 minutes to reach 34° C. Approximately 250 mL portions of the 34°C. serum (total volume about 1 liter) were transferred to one-literErlenmeyer flasks and tightly capped with aluminum foil. These wereincubated for 20-30 minutes (preferably 30 minutes) in the 34° C.orbital shaker water bath at a medium-high rotation speed. Thereafter,25 mL of the charcoal/dextran suspension was added to each flask. Thecharcoal-dextran suspension was stirred at room temperature whileremoving the 25 mL aliquot. The final charcoal concentration in eachflask was 10 mg/mL, and the final concentration of dextran was 1 mg/mL.After addition of the charcoal-dextran mixture to each flask, theextraction mixtures were shaken at 140-160 RPM at 34° C. for two hours.After this, the mixture was cooled on ice and the charcoal removed bycentrifugation at 10,000×g for about 60 minutes at room temperature. Insome preparations the temperature of the mixture gradually warmed toabout 40° C. during centrifugation. The supernatants were pooled in atwo-liter beaker and 275 mL portions of the supernatant (serum)transferred to fresh one-liter Erlenmeyer flasks. These were thenincubated in the orbital shaker water bath at 34° C. for 20-30 minutes(preferably 30 minutes) to re-equilibrate the temperature. A secondextraction was done by addition of a fresh aliquot (about 14 mL) of thecharcoal-dextran suspension. This re-extraction mixture was incubatedwith shaking for another 2 hours at 34° C. The final charcoalconcentration during this extraction was about 5 mg/mL. Afterward thebulk of the charcoal was removed by centrifugation, as before. In somepreparations the temperature of the mixture reached about 41° C.,without harming the quality of the serum. The supernatants werecollected into a two-liter beaker and filtered through 5 μm porediameter filters to remove residual charcoal. If it was considerednecessary for particular preparations that still contained residualcharcoal, (for example, due to charcoal darkening serum) the serum wasalso filtered with 0.45 μm pore diameterMillipore filters. Thesefiltrations were done with plastic reusable filter holders and lightvacuum. The steroid hormone depleted serum was then sterilized using 0.2μm pore diameter filters. After sterilization, aliquots of about 26 mLwere dispensed into sterile glass (50 mL) bottles or sterile 50 mLpolypropylene tubes and stored frozen at −17° C. Although 34° C. ispreferred in the above-described regime, and provides the best results,satisfactory depletion of steroid hormones can be obtained over thetemperature range of about 30 to 37° C. The 2 hour incubation times forthe extraction and re-extraction mixture (at 34° C.) are preferred, buta time range of 30 minute to 3 hours could also be used with success,employing longer incubation times for the lower temperatures within the30-37° C. range. A±25% variation in the charcoal concentration used foreach extraction had no detrimental effects on the final product.

B. Amberlite™ XAD™-4 Resin Treatment. In a different procedure carriedout to free corticosteroids binding globulin (CBG) of storage cortisol,XAD resin has been used to remove the steroid by incubation for 5 hrs atroom temperature (A. M. Nakhla, et al. (1988) Biochem. Biophys. Res.Commun. 153, 1012-1018). Described as such, this method removed onlyabout 80% of cortisol from the purified protein. Careful application ofthat method failed to yield serum suitable for the purpose of thisstudy. As an alternative to preparing steroid hormone depleted serum bycharcoal-dextran extraction, horse serum was treated by incubation withAmberlite™ XAD™-4 nonionic polymeric absorbent (Aldrich, Catalog. No.21, 648-8; or Sigma. Catalog No. XAD-4 37380-42-0). Specifically, a 500mL bottle of horse serum was thawed at 37° C. and divided into 250 mLportions that were each in a one-liter Erlenmeyer flask. To each flaskwas added 25 grams of moist XAD-4™ resin. The mixtures of serum andresin were then incubated with shaking in a rotary Labline OrbitalShaker water bath at 34° C. at about two-thirds of the maximum rate for24 hours (speed adjusted to control foaming). This extraction can bedone at temperatures from 30° C. to 37° C. At 30° C., the extractionrequires 24 to 36 hours. At 37° C., it requires 18-24 hours to becomplete. The 34° C. and 37° C. procedures are preferred. Each flask wastightly capped with aluminum foil and taped. After 24 hrs, the resin isallowed to settle by gravity, the supernatant decanted, and then vacuumfiltered using a glass fiber filter in a Buchner funnel. The resultingserum was filter sterilized using 0.2 μm pore filter units. Aliquots ofabout 26 mL were frozen at −17° C. in 50 mL sterile bottles or 50 mLpolypropylene tubes.

Discussion of Example 2. Each of the methods presented have advantages,depending on the particular needs and desires of the user. The scaleprocedures described are useful to prepared sufficient serum for testingof plasma or bodily fluids samples for inhibitors and for hormoneactivities or anti-hormone activities or evaluation of toxicity ofcompounds in cell culture assays. To ensure uniformity, large batches ofthe serum can be prepared, if desired. The charcoal method describedabove is readily applicable to one to five liter volumes of serum perpreparation. With use of moderate numbers of test samples or ≦50 mL pertest substance, this is an adequate supply. To prepare larger volumes ofserum (i.e. ≧20 liters) for extensive testing programs or commercialapplications, the charcoal-dextran methods will preferably employindustrial filtration or other separation equipment to remove the carbonafter each extraction. The XAD-4™ resin method as presented is adaptableto one to five liters for testing purposes. For industrial applications,where ≧20 to 100 liter batches are customarily required, the resinmethod is preferred because of the need for only one separation afterextraction. However, where “foaming” of the serum protein is to beavoided completely, charcoal extraction is superior. The materials costfor charcoal-dextran has an advantage when economy is a majorconsideration. It is less expensive than XAD-4™ resin on a per literbasis, although the resin is commercially available at low cost whenpurchased in large amounts (i.e. ≧50-100 kilograms). XAD-4™ resin methodis highly adaptable to small clinically derived samples of plasma orother bodily fluids.

This 34° C. method has been used to prepare CDE human serum, porcineserum, rat serum, hamster serum, ovine serum, fetal bovine serum, newborn bovine serum (0 to 10 days old), young donor bovine serum (10 daysto 6 moths old) young adult bovine serum (300 to 900 lbs), fetal horseserum, chicken serum, turkey serum, dog serum, goat serum, rabbit serumand monkey serum. Subsequent Examples demonstrate how these strippedsera are preferably employed. The results demonstrate the broad utilityof the method of preparing charcoal-dextran extracted serum for testingof cell lines from many species using homologous serum assays. Fromthese results it can also be readily appreciated that these methods areapplicable not only to testing of human plasma/serum, but also toveterinary medicine samples or compounds of significance to domesticanimals, as well as any application where a steroid hormone strippedserum is used. For example, in the diagnosis, prevention/risk managementor therapy of mucosal origin cancers.

Example 3 MTW9/PL2 Rat Mammary Tumor Cells

This Example describes a sensitive in vitro model assay system fordetecting and measuring steroid hormone responsive cell growth.

The MTW9/PL2 Rat Mammary Tumor Cell Line. The properties of the MTW9/PL2have been summarized (Moreno-Cuevas J E and Sirbasku D A In Vitro CellDev Biol 36, 410-427). The MTW9/PL cell line was established by ourlaboratory in culture from the carcinogen-induced hormone-responsiveMT-W9A rat mammary tumor of a W/Fu rat. This tumor formed estrogen,androgen and progesterone responsive tumors in W/Fu rats (Sirbasku D A(1978) Cancer Res 38, 1154-1165). It was later used to derive theMTW9/PL2 cell population that was also estrogen-responsive in vivo(Danielpour D et al. (1988) In Vitro Cell Dev Biol 24, 42-52). In serumsupplemented culture conditions the MTW9/PL2 cells demonstrate 80-foldsteroid hormone growth responses. All sera used were steroidhormone-depleted by charcoal-dextran treatment at 34° C. The studieswere done with horse serum as well as serum from other mammalianspecies. The growth of the MTW9/PL2 cells was biphasic in response tohormone-depleted serum. Concentrations of ≦5% (v/v) promoted optimumgrowth. Above this concentration, serum was inhibitory.Concentrations≧40% (v/v) inhibited growth altogether. Addition of1.0×10⁻¹³ to 1.0×10⁻⁸ M 17-estradiol (E₂) reversed the inhibitioncompletely. At 1.0×10⁻⁸ M, E₁, E₃ and DES promoted growth as well as E₂.Testosterone and DHT promoted growth only at 10⁻⁷ M. Progesterone waseffective at 10⁻⁶M. Cortisol was ineffective. Labeled hormone bindinganalysis and Western immunoblotting documented that MTW9/PL2 cells hadestrogen and progesterone receptors but not androgen or cortisolreceptors. Estrogen treatment of MTW9/PL2 cells induced a concentrationand time dependent increase in progesterone receptors. It was concludedthat (1) the MTW9/PL2 population is the first highly steroid hormoneresponsive rat mammary tumor cell line to be established in culture froma carcinogen induced tumor and (2) sera from a number of speciesincluding horse, rat and human contain an inhibitor which mediatesestrogen sensitive MTW9/PL2 cell growth in culture.

Estrogenic Effects with MTW9/PL2 Rat Mammary Tumor Cells in CulturesSupplemented with CDE-horse Serum. Unless otherwise stated, referencesin this and the following Examples to “CDE-horse serum” refer to the 34°C. charcoal-dextran extraction process described in above. The MTW9/PL2cells were assayed for E₂ responsiveness in cultures supplemented withincreasing concentrations of CDE-horse serum (FIG. 1A).Concentrations≦5% (v/v) promoted growth. Typically within seven dayscell numbers increased from 6,000 per dish to more than 200,000 in 2 to5% serum. This most likely resulted from stimulation by serum-bornegrowth factors as well as the mitogenic effect of transferrin(Danielpour D et al (1988) In Vitro Cell Dev Biol 24, 42-52; Riss T Land Sirbasku D A (1987) In Vitro Cell Dev Biol 23, 841-849; Riss T L etal. (1986) J Tissue Culture Methods 10, 133-150). As serumconcentrations exceeded 5% (v/v), the effects of the growth promoterswere counteracted by a serum-borne inhibitor(s). At serum concentrationsof 30 to 50% (v/v), growth was completely inhibited. Usually only seeddensity cell numbers were found after seven days in cultures containing50% (v/v) CDE-horse serum. In contrast, the presence of 1.0×10⁻⁸ M E₂completely reversed the serum dependent inhibition. In culturessupplemented with 20 to 50% (v/v) CDE-serum plus 1.0×10⁻⁸ M E₂, cellnumbers were 400,000 per dish. Logarithmic quantifying of cell growthwas done by converting the cell number data in FIG. 1A into CPD. A plotof these values is shown in FIG. 1B. The estrogenic effect is alsopresented. In FIG. 1B, the difference was maximum at 30% (v/v) CDE-horseserum. It was a 6.14 CPD or a 70-fold (i.e. 2^(CPD) or 2^(6.14))increase in cell numbers in response to E₂. In randomly selectedreplicate experiments (N=9) done over a two-year period with differentpreparations of CDE-horse serum, the average maximum estrogen effect±SEMwas 6.43 CPD±0.49 (range 5.63 to 7.22). This was an 86-fold (2^(6.43))estrogenic effect. The modal concentration of serum that promotedmaximum E₂ effects was 40% (range 20 to 50%).

Estrogen Reversibility of the Growth Inhibition Caused by CDE-horseSerum. It was examined whether inhibition caused by CDE-horse serum wasreversible even after several days in culture (FIG. 2). The MTW9/PL2cells were seeded into medium containing 50% (v/v) CDE-horse without E₂and cell numbers monitored daily. Growth ceased within 48 hours;thereafter cell numbers remained static. In parallel cultures, additionof E₂ on days two, four, and six after seeding caused resumption ofgrowth (after a lag period) at nearly the same rate as cultures thatreceived hormone at the time of inoculation. These results show that thecells survived in the presence of the inhibitor without E₂ for at leastsix days. As described in a later Example, longer exposure to thepurified inhibitors was cytotoxic and suggested therapeutic value.

Storage Stability of CDE-horse Serum. In Table 3, the effect of storagetemperature on the estrogen mediating activity of CDE-horse serum isshown. The assays were done with MTW9/PL2 cells as shown in FIGS. 1A and1B. Stability was assessed by four criteria: (i) the concentration ofserum needed to give an estrogenic effect of 2.5 CPD (i.e. ED_(2.5)),(ii) the percent serum needed for the maximum estrogenic effect, and themagnitude of the estrogenic effects (CPD) at (iii) 20% and (iv) 30%serum. CDE-horse serum was stable at 23° C. for three weeks without lossof activity as assessed by all four criteria. Storage at 4° C. wasdetrimental within 24 days as measured by the CPD at 20% and 30% (v/v)serum concentrations. Longer storage at 4° C. was not advisable. Storageat −17° C. was most effective; the activity was unchanged even after 90days. In experiments not shown, repeated freeze-thaw cycles caused anappreciable loss of inhibitor activity. The results in Table 3 show thatserum stored frozen has utility for long periods and therefore providesa stable supply for testing of clinically relevant samples. Also, it isclear that clinical samples to be assayed for inhibitor can be storedfor a few days at room temperature without damage.

TABLE 3 Summary of the Effects of Serum Storage Temperature on Activity.% Serum needed Maximum E₂ CPD at Days for 2.5 CPD Induced CPDs 20% of(ED_(2.5)) of E₂ (% serum, v/v, for (v/v) CPD at 30% Storage Inducedgrowth the maximum) serum (v/v) serum Storage at 23° C. 1 2.1 4.9 (10%)5.0 3.2 3 5.2 5.4 (20%) 6.2 5.2 6 5.0 4.2 (10%) 3.5 0.9 14 2.9 6.0 (10%)4.3 2.6 23 4.0 6.3 (10%) 3.9 2.5 Storage at 4° C. 1 1.8 5.9 (10%) 4.94.0 7 6.8 5.7 (20%) 6.4 5.4 15 3.8 4.1 (30%) 5.5 4.2 24 5.3 5.3 (10%)1.0 2.8 44 3.0 4.8 (5%) 0.04 0.26 55 2.2 5.0 (5%) 0.00 0.24 90 >50 2.1(5%) 0.30 0.40 Storage at −17° C. 1 2.6 5.2 (10%) 5.0 3.1 7 4.0 5.8(30%) 6.8 5.8 44 3.3 5.8 (20%) 6.0 5.4 90 6.1 5.2 (30%) 6.2 5.9

Dose-Response Effects of Steroid Hormones in CDE-horse Serum. The doseeffects of a number of steroid hormones were evaluated with MTW9/PL2cells in medium containing 50% (v/v) CDE-horse serum. The results of oneof these studies (N=3) are presented in FIG. 3. Estrogens were the mosteffective mitogens. Their order of potency was E₂>E₁>E₃. This relativepotency was expected based on the affinities of these steroids for theestrogen receptors of other target tissues (Clark J H and Markaverich BM (1983) Pharmacol Ther 21, 429-453). The cell numbers in dishescontaining 1.0×10⁻¹³ M E₂ were 32-fold (p<0.01) higher than in disheswithout the hormone. Concentrations of 1.0×10⁻¹² to 1.0×10⁻¹¹ M E₂promoted 6.73 CPD that was a 110-fold estrogenic effect in seven days.The ED₅₀ of E₂ was about 0.5 to 1.0×10⁻¹² M. Using E₁ and E₃, optimumgrowth was achieved at 1.0×10⁻⁹ and 1.0×10⁻⁸ M, respectively. Inexperiments not shown, the mitogenic potency of the synthetic estrogenDES was assessed. At 1.0×10⁻⁸ M, it caused the same growth as saturatingconcentrations of the natural estrogens. The DES effect was 6.98 CPD inseven days that was a 126-fold (2^(6.98)) increase in cell number. Thenext most potent hormone was DHT. It caused significant (p<0.05) growthat supraphysiologic concentrations≧1.0×10⁻⁸ M. Progesterone also causedsignificant growth, but only at supraphysiologicalconcentrations≧1.0×10⁻⁷ M. Cortisol was ineffective at concentrations upto 1.0×10⁻⁶M.

Estrogen Mitogenic Effects with MTW9/PL2 cells in CDE-serum from SeveralSpecies. Serum from species other than horse were examined to determinethey also possessed estrogen reversible inhibitory activity with ratMTW9/PL2 cells. These experiments are shown in FIG. 4. All of the seratested were charcoal dextran extracted at 34° C. CDE-porcine (FIG. 4A),and CDE-human serum (FIG. 4B) showed patterns nearly identical to thatof CDE-horse serum. The maximum estrogenic effects with both sera weresix to seven CPD (N=3). CDE-rat serum also showed the same pattern ofestrogen reversible growth inhibition (FIG. 4C). CDE-ovine serum showedestrogen reversible inhibition equivalent to CDE rat serum (data notshown). With serum from rats, the maximum estrogenic effect was four tofive CPD (N=4). CDE-bovine serum (adult donor herd) displayed the samepattern of activity as other sera (FIG. 4D). CDE-fetal bovine serumshowed a different pattern (FIG. 4E). Even at 40% (v/v), there was noinhibition. With some batches of this serum, there was no inhibitioneven at 50% (N=2). With others (N=2), inhibition was found. In theseexperiments, the estrogenic effects reached three to four CPD in 50%(v/v) CDE-serum. Even with this variability, fetal bovine serum has lessactivity than the serum from the adults of this species. The assays withCDE-fetal horse serum (N=3) showed inhibition at 50% (v/v) that was notreversible by 10 nM E₂ (FIG. 4F).

Discussion of Example 3

The MTW9/PL2 Cell Line as a Unique Rodent Test System. The present studyshows very clearly that (ER⁺) MTW9/PL2 cells are estrogen growthsensitive in culture and applicable to testing of serum or bodily fluidinhibitors or sex steroids in such preparations. The estrogen receptorcontent and estrogen affinity characteristics of the MTW9/PL2 cellsindicate appropriate stability for commercial applications. The MTW9/PL2population is the first highly steroid hormone-responsive rat mammarytumor cell line to be established in culture from a carcinogen-inducedtumor”. As a direct consequence of the information provided above, thiscell line is a unique and valuable asset for combination in vitro and invivo modalities to be applied to clinically and commercially significantcompounds or preparations and for the assay of the inhibitor content orhormone or anti-hormone activities.

Technical Conditions for Demonstrating Estrogen Responsiveness inCulture and Evidence for a Serum-borne Inhibitor. Conditions that permitthe observation of very large magnitude estrogen mitogenic effects withthe permanent MTW9/PL2 cell line in culture are defined herein. Asmentioned in the Background of the Invention, most existing rat mammarytumor cell lines are not suitable for use in evaluating hormoneresponsiveness in vivo because they are derived from outbred animals.This problem was overcome by developing the MTW9/PL2 rat mammary tumorcell line from a carcinogen-induced hormone responsive tumor (i.e. theMT-W9A tumor), first induced and transplanted in an inbred W/Fu rat asdescribed (MacLeod R M et al. (1964) Cancer Res 75, 249-258). TheMTW9/PL2 cells form hormone responsive tumors when implanted in theserats (Sirbasku D A (1978) Cancer Res 38, 1154-1165; Danielpour D andSirbasku D A (1984) In Vitro 20, 975-980; Riss T L et al. (1986) JTissue Culture Methods 10, 133-150). In culture, the MTW9/PL2 cellsshowed the same hormone responsiveness expected of rat and human breastepithelial cells, as shown herein and subsequently reported(Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36,410-427; Sirbasku D A and Moreno-Cuevas J E (2000) In Vitro Cell DevBiol 36, 428-446; Moreno-Cuevas J E and Sirbasku D A (2000) In VitroCell Dev Biol 36, 447-464).

The effects of the steroid hormones in culture were the same asdescribed for the growth of the original MT-W9A tumor in W/Fu rats(MacLeod et al. (1964) Endocrinology 75, 249-258) and tumor formation bythe parental MTW9/PL cell line in this same strain of rats (Sirbasku D A(1978) Cancer Res 38, 1154-1165). The present embodiment is the firstestablished cell line derived from a carcinogen induced rat mammarytumor that continues to show large magnitude growth responses toestrogens, progesterone and androgens even after extended periods inculture, preferably when cultured under the conditions disclosed herein.Thyroid hormone responsiveness has also been demonstrated for MTW9/PLcells (Leland F E et al. (1981) In: Functionally Differentiated CellLines, Sato G, ed, Alan Liss, New York, pp 1-46). Of the other importanthormones known to influence the growth of the original MT-W9A tumor,only prolactin remains to be investigated. Prolactin is not mitogenicfor the parental MTW9/PL cells under serum-free defined conditions(Danielpour D et al. (1988) In Vitro Cell Dev Biol 24, 42-52).Continuing investigations are directed toward evaluating the possibilitythat prolactin also reverses the effects of the serum-borne inhibitor orotherwise acts as a cytokine to influence the production ofimmunoglobulins in breast and other mucosal tissues. The development ofthis cell line now permits not only sensitive steroid hormone growthanalysis in culture, but also direct comparisons to the effectiveness ofthe same test substances in animals. No other such rat mammary system iscurrently available.

MTW9/PL2 Receptor Not Lost in Culture. The present results showing anaverage 86-fold MTW9/PL2 cell number increase in seven days in responseto physiological concentrations of E₂ have several important technicalimplications. Most notably, they contradict many earlier explanationsfor why estrogen stimulated cell growth has been difficult todemonstrate in culture. Originally, the lack of estrogenic effects inculture was thought to be due to a dedifferentiation of cells thatresulted in a loss of functional receptors or some other aberration thatdisrupted the growth response. In light of the present Disclosure, thisexplanation now seems very unlikely. The present results show thepresence of similar levels of estrogen receptors in both the originalMTW9/PL cell line reported in 1982 and the current MTW9/PL2 cells.Analyses made by others showing estrogen receptors in established celllines in culture (Horwitz K B et al. (1978) Cancer Res 38, 2434-2437;Haug E (1976) Endocrinology 104, 429-437; Soto A M et al. (1988) CancerRes 48, 3676-3680; Keydar I et al. (1979) Eur J Cancer 15, 659-670;Engel L W et al. (1978) Cancer Res 38, 3352-3364) also mitigate againstthis explanation. Furthermore, the estrogen receptors of the MCF-7 cellswere functional based on the demonstration of estrogen inducibility ofthe progesterone receptor (Horwitz K B and McGuire W L (1978) J BiolChem 253, 2223-2228). As with the human breast cancer cells, theMTW9/PL2 line was also significantly estrogen responsive by thiscriterion. When all of the available data is considered in light of thepresently disclosed observations, the notion that long-term culturenecessarily leads to loss of functional estrogen receptors is laid torest. A major advantage of the MTW9/PL2 line is its long-term stabilitypermitting series analyses over long periods of time without concern forcell property changes.

Prolonged Steroid Hormone Retention by Culture Cells. It has beensuggested that prolonged retention of estrogens might be the reason fora lack of responsiveness of target cells in culture (Strobl J S andLippman M E (1979) Cancer Res 39, 3319-3327). Investigators havereported that the half-life of loss of specifically bound ³H-E₂ fromMCF-7 cells was about 24 hours at 37° C. (Strobl J S and Lippman M E(1979) Cancer Res 39, 3319-3327). Cells from stock cultures grown inuntreated/steroid hormone containing serum were proposed to retainstimulating levels of estrogens. Even several washes over 78 hours didnot attenuate the problem (Strobl J S and Lippman M E (1979) Cancer Res39, 3319-3327). Conversely, the studies herein did not identify thisproblem. All the assays reported here were done with cells takendirectly from cultures grown in steroid hormone containing serum (e.g.fetal bovine serum). After trypsinization of the MTW9/PL2 cells fromstock culture, only three careful washes with serum-free D-MEM/F-12 wereperformed before initiating the growth assays. The results in FIG. 3show clearly that 1.0×10⁻¹² M E₂ caused significant MTW9/PL2 cellgrowth. Also, the results in FIG. 2 show that MTW9/PL2 cells ceaseproliferation within 48 hours of starting a growth assay. Theseobservations either support the conclusion that prolonged steroidhormone retention by cells is not as serious an issue as first proposedor are evidence that the technical processes described herein to preparecells for assays have eliminated this problem. With regard to thepresent investigation, all cell lines studied showed this same propertywhen prepared by the same technical process for growth assays.

Merits of Charcoal Extraction. Other investigators have challenged theuse of charcoal extraction to deplete serum of steroid hormones. It hasbeen stated that this procedure absorbs or otherwise alters serum tomake it ineffective (Amara J F and Dannies P S (1983) Endocrinology 112,1141-1143; Wiese T E et al. (1992) In Vitro Cell Dev Biol 28A, 595-602).To counter this problem, either individual lots of untreated serum wereused to seek estrogenic effects (Wiese T E et al. (1992) In Vitro CellDev Biol 28A, 595-602), or serum was prepared from animals afterendocrine ablation surgery (Amara I F and Dannies P S (1983)Endocrinology 112, 1141-1143). One of the best examples of use ofsurgically depleted serum came from the study of the GH₄C₁ rat pituitarycells (Amara J F and Dannies P S (1983) Endocrinology 112, 1141-1143).They were highly E₂ responsive in medium supplemented with the serumfrom a gelded horse (Amara J F and Dannies P S (1983) Endocrinology 112,1141-1143). However, experience with serum derived by these methods hasnot been as positive. For example, this issue was investigated in 1976with the related GH₃C₁₄ rat pituitary tumor cell line (Kirkland W L etal. (1976) J Natl Cancer Inst 56, 1159-1164), and found that serum fromovariectomized sheep or adrenalectomized and ovariectomized sheep didnot support estrogen effects. Furthermore, unextracted sera fromdifferent species can at times support limited estrogenic effects.However, the estrogenic effects are of lower magnitude than those in theCDE-serum described herein. The results are so variable that theytypically exclude use as a clinical testing assay. Based on theobservation that CDE-serum from a number of species was very effective,it seems highly unlikely that the now-disclosed preferred 34° C.procedure is deleterious. However, it is clear from other studies thatthe 56° C. charcoal method caused a temperature dependent loss of theinhibitor (data not shown). The presently described CDE-serum providesgreater consistency and reproducibility than the other proposedapproaches (Amara J F and Dannies P S (1983) Endocrinology 112,1141-1143; Wiese T E et al. (1992) In Vitro Cell Dev Biol 28A, 595-602).Another advantage is that these results do not dependent significantlyon the lot of serum purchased. Furthermore, CDE-serum consistentlyprovides larger magnitude estrogenic effects than serum obtained byeither of the other approaches discussed above.

Steroid Hormone Conjugates are Non-problematic. While charcoal treatmentcan be expected to remove the major classes of steroid hormones fromserum, there is a question about its effect on the more soluble andpotentially active conjugates. It has been reported that hydrolysis ofestrogen sulfates provided free estrogens in human breast cancer cellcultures (Vignon F et al. (1980) Endocrinology 106, 1079-1086). Thisabrogated the effects of exogenous E₂. Although the previousinvestigations did not address estradiol sulfate, it was shown that morethan 95% of estrone sulfate and estradiol glucuronide were removed fromserum by a single 56° C. charcoal extraction (Sirbasku D A and KirklandW L (1976) Endocrinology 98, 1260-1272). Additionally, in previousstudies MTW9/PL cells were incubated with tritium labeled estradiolglucuronide for up to 24 hours under cell culture conditions and foundno organic solvent extractable free steroid. Both past and currentresults indicate that the impact of the estrogen conjugates has beenoverestimated. In the present study, no precautions were taken to removethe conjugated forms of estrogens from any of the sera tested. Despitethis, it was found that many different types of serum were effectiveafter charcoal extraction at 34° C. Thus, it is concluded that removalof steroid conjugates by digestion or any procedure beyond charcoaltreatment is unnecessary. This is a further advantage of the new 34° C.method because the additional treatment to remove the steroid conjugatescould be prohibitively expensive for larger scale production than a fewliters, and could potentially introduce undesirable effects in theserum.

Plastic Product Use for Cell Culture. The present studies were done withplastic ware made of polystyrene. Plastic is manufactured usingalkylphenols (Platt A E (1978) In: Encyclopedia of Chemical Technology,Kirk R E, Othmer D F, eds, 3^(rd) Edition, Volume 26, Wiley, New York,pp 801-847). One of these compounds, p-nonyl-phenol, has been reportedto be estrogenic for MCF-7 cells in culture (Soto A M et al. (1991)Environ Health Perspect 92, 167-173). This xenobiotic most likely ispresent in the cultures used in these studies. No precautions were takento exclude compounds leaching from plastic. In fact, the bioassayprocedures herein are done with polystyrene plastic ware and culturedishes almost exclusively. If there had been a significant contaminationof the medium by such compounds, the estrogenic effects reported in thisstudy should not have been seen or should have been markedly attenuated.An advantage of the assay systems described herein is that they have noneed for expensive and or exotic substitutes for the common plastic wareused in cell culture laboratories to conduct bioassays. Also, theCDE-serum can be stored and shipped for commercial use in conventionalplastic containers without concern for creation of plastic-inducedartifacts. Clinical samples for assay can also be stored and shipped incommon plastic ware.

Example 4 Estrogen Responsive Growth of Additional Rodent and Human CellLines in 34° C. Charcoal-Dextran Extracted Horse and Human Serum

In addition to the above-described studies using the MTW9/PL2 ratmammary tumor cell line, several other cell lines were employed todefine the conditions for demonstrating estrogen and androgen responsivecell growth. Established cell lines from a number of different steroidhormone target tissues were selected for growth regulation analysisunder those defined conditions. Additional model cell growth assays formeasuring steroid hormone responsive cell growth are described.

Estrogen Mitogenic Effects with Established ER⁺ Rodent Tumor and HumanCarcinoma Cells in CDE-horse Serum. In the first study of this series,the three GH rat pituitary tumor cell lines were examined for estrogeniceffects in CDE-horse serum. This was considered important in light oftheir clear responsiveness to many hormones (Tashjian A H Jr (1979)Methods Enzymol 58, 527-535). Furthermore, these cells are from a tissuethat grows coordinately with mammary tissue in castrated ratsadministered exogenous estrogens. As described above, this suggested acommon regulation mechanism. FIG. 5 shows an estrogenic effect≧5 CPDwith GH₄C₁ cells in 10 days. The results with GH₃ and GH₁ cells rangedbetween 4.0 and 5.2 CPD in 10 to 14 day assays (data not shown). Thesame progressive estrogen reversible CDE-serum inhibition wasdemonstrable with both rat mammary and rat pituitary tumor cells inCDE-horse serum. To confirm the effectiveness CDE-horse serum with humancells, the ZR-75-1 breast cancer line was selected because of previousattempts to demonstrate its estrogen responsiveness in culture (AllegraJ C and Lippman M E (1978) Cancer Res 38, 3823-3829; Darbre P D et al.(1984) Cancer Res 44, 2790-2793; Darbre P et al. (1983) Cancer Res 43,349-355). The ZR-75-1 cells showed the same CDE-serum caused estrogenreversible inhibition as seen with rodent cell lines in this serum. In14 days, there was a 3.65 CPD 12.5-fold) estrogenic effect (FIG. 6).This was a greater response than recorded in the ZR-75-1 cell studiescited above. Of all of the cell lines studied, the MCF-7A was the leastestrogen responsive even in 50% CDE-horse (FIG. 7). The estrogeniceffect was 2.65 CPD in 10-12 days. This was still significant (p<0.01)as a 2^(2.65) or 6.3-fold increase in cell number caused by estrogen.The present serum-derived inhibitor exhibited biological activityexactly opposite the estrogen reversible inhibitors described by M Tanjiet al. (Tanji M et al. (2000) Anticancer Res. 20, 2779-2783; Tanji M etal. (2000) Anticancer Res. 20, 2785-2789).

Additional Cell Lines Evaluated. Evidence is presented herein that theMCF-7K, T47D, LNCaP, and H301 cells are highly sex steroid hormoneresponsive in CDE-horse serum.

Kinetics of Estrogen Responsive Growth in CDE Serum Containing Medium.In the experiments presented in FIGS. 8A and 8B, ER⁺ cell growth wasmeasured daily for 15 days to determine cell growth kinetics±E₂. Theresults with the T47D line are presented as characteristic of humancells. When evaluated in medium with partially inhibitory 20% (v/v) CDEhorse serum, the effect of E₂ on cell number increase was not apparentuntil after 4 days (FIG. 8A). Increasing the concentration of CDE serumto 50% (v/v) further delayed the effect of E₂ (FIG. 8B). Clearly,whatever mechanism is proposed for the action of the steroid hormone, ittakes a significant period to reverse the effects of the inhibitor. Thisprocess cannot be simply due to a rapid effect on transcription causedby steroid hormones. The interaction of ³H-E₂ with intracellularestrogen receptors saturates in ≦1 hour at 37° C. (Horwitz K B andMcGuire W L (1978) J Biol Chem 253, 8185-8191; MacIndoe H I et al.(1982) Steroids 39, 245-258; Moreno-Cuevas J E and Sirbasku D A (2000)In Vitro Cell Dev Biol 36, 410-427), while de novo hormone inducedprotein synthesis requires only a few hours (Beato M (1989) Cell 56,335-344). Based on a growth lag of ≧4 days, it is likely that steroidhormones initiate a cascade of signaling events that are more complexthan recognized today. To demonstrate that the lag period was related tothe inhibitor, T47D growth was monitored daily in D-MEM/F-12supplemented with 10% (v/v) fetal bovine serum (FIG. 8A). Thisconcentration of fetal bovine serum shows no inhibitor (Moreno-Cuevas JE and Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 410-427). Cellgrowth in medium with fetal bovine serum showed at most a one or two daylag period.

Effect of CDE-human Serum on Estrogen Responsive Cell Growth. The nextstudy examined whether human serum was a source of inhibitor for steroidhormone sensitive cell lines from different species and tissues. Theresults confirm that CDE-human serum contains approximately the samelevel of inhibitor as CDE-horse serum. Results are shown with T47D humanbreast cancer cells (FIG. 9A), LNCaP human prostatic carcinoma cells(FIG. 9B), MTW9/PL2 rat mammary tumor cells (FIG. 9C), two GH ratpituitary tumor cell lines (FIGS. 9D and 9E), and the Syrian hamsterH301 kidney tumor cells (FIG. 9F). All lines showed the same biphasicresponse to CDE-human serum. Low concentrations (i.e. ≦10%) promotedgrowth whereas higher concentrations (i.e. ≧20%) progressively inhibitedgrowth. Only the absolute magnitudes of the estrogenic effects varied.Replicate assays with MCF-7A, MCF-7K and ZR-75-1 cells gave the sameoutcomes (data not shown). The experiments reported thus far hereinsupport the conclusion that the inhibitor is ubiquitous in mammals andis not species specific, also subsequently reported (Sirbasku D A andMoreno-Cuevas (2000) In Vitro Cell Dev Biol 36, 428-446).

Dose-response Effects of Steroid Hormones with Human Breast Cancer Cellsin CDE Serum. The studies presented thus far have assessed estrogeneffects using 10 nM E₂. Although 10 nM saturates growth, it is decidedlyat the high boundary of physiological. It is important to note thatcirculating estrogens in non-pregnant females are generally thought tobe in the range of 10⁻⁸ to 10⁻¹⁰ M (Clark J H et al. In: WilliamsTextbook of Endocrinology (1992), Saunders, Philadelphia, pp 35-90).Tissue concentrations are generally conceded to be lower due to SHBGthat reduces the “free” or “active” form of sex steroid hormones (RosnerW (1990) Endocr Rev 11, 80-91). The next studies with T47D cellsdetermined the effective concentration ranges for the three most commonestrogens and compared these to non-estrogen steroid hormones. FIG. 10shows an analysis with T47D cells in D-MEM/F-12 containing 50% (v/v) CDEhorse serum for 14 days. Estrogens were the only physiologicallyrelevant activators of T47D growth. As expected from previous studieswith breast cancer cells (Lippman M E et al. (1977) Cancer Res 37,1901-1907; Jozan S et al. (1979) J Steroid Biochem 10, 341-342;Katenellenbogen B S (1980) Annu Rev Physiol 42, 17-35) and otherestrogen target tissues (Clark J H and Markaverich B M (1983) PharmacolTher 21, 429-453), their order of effectiveness was E₂>E₁>E₃. E₂ causedsignificant (p<0.05) growth when present at 1.0×10⁻¹⁴ M and optimumgrowth at 1.0×10⁻¹⁰ M. Higher concentrations were not inhibitory. TheED₅₀ concentration of E₂ was <1.0×10⁻¹³ M. It is noteworthy that even E₃was remarkably potent. Others also had commented that E₃ was more potentthan expected (Lippman M E et al. (1977) Cancer Res 37, 1901-1907). Thisobservation may have special significance because breast cancers thatappear during pregnancy can be particularly life threatening. Humanmaternal plasma has greatly elevated levels of E₃ during the lasttrimester of pregnancy. Testosterone and DHT promoted growth but only atsupraphysiological concentrations (FIG. 10). Other investigators havesuggested that supraphysiological concentrations of androgens actthrough the ER of human breast cancer cells (Zava D T and McGuire W L(1978) Science (Wash DC) 199, 787-788). However, another group hasreported no effect of androgens on human breast cancer cellproliferation (Soto A M and Sonnenschein C (1985) J Steroid Biochem 23,87-94). In the present study, progesterone and cortisol were completelyineffective with T47D cells (FIG. 10). Others have also reportednegative results with these hormones and human breast cancer cells(Schatz R W (1985) J Cell Physiol 124, 386-390; Soto A M andSonnenschein C (1985) J Steroid Biochem 23, 87-94). The data presentedin this Disclosure support the conclusion that the new CDE serum cultureconditions yield physiologically relevant information.

Dose-response Effects of Steroid Hormones with Rat Pituitary Tumor Cellsin CDE Serum. The GH family of related cell lines responds to a numberof different classes of hormones (Amara J F and Dannies P S (1983)Endocrinology 112, 1141-1143; Tashjian A H Jr et al. (1970) J Cell Biol47, 61-70; Tashjian A H Jr (1979) Methods Enzymol 58, 527-535; Haug E(1979) Endocrinology 104, 429437; Schonbrunn A et al. (1980) J Cell Biol85, 786-797; Sorrentino J M et al. (1976) J Natl Cancer Inst 56,1159-1164; Ramsdell J S (1991) Endocrinology 128, 1981-1990; Hayashi Iet al. (1978) In Vitro 14, 23-30; Faivre-Bauman A et al. (1975) BiochemBiophys Res Commun 67, 50-57). These cells also form steroid hormoneresponsive tumors in W/Fu rats (Sorrentino J M et al. (1976) J NatlCancer Inst 56, 1149-1154). The GH₄C₁ strain was selected as an examplefor this next study because of its marked E₂ responsiveness in culture(Amara J F and Dannies P S (1983) Endocrinology 112, 1141-1143; SirbaskuD A and Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446;Sato H et al. (1991) In Vitro Cell Dev Biol 27A, 599-602) and estrogenrequirement for tumor formation in rats (Riss T L and Sirbasku D A(1989) In Vitro Cell Dev Biol 25, 136-142). The dose-response effect ofsteroid hormones with GH₄C₁ rat pituitary tumor cells in 50% CDE-horseserum was analyzed next. FIG. 11 shows the results of these experiments.All three major estrogens promoted growth. The potencies of E₂ and E₁were equivalent whereas E₃ was substantially less effective. Even atsupraphysiologic concentrations, E₃ did not promote the saturationdensities seen with E₂ and E₁. The lowest concentration of E₂ and E₁that gave significant (p<0.05) growth was 1.0×10⁻¹²M. The ED₅₀ of E₂ was≦1.0×10⁻¹¹ M. Optimum growth required supraphysiological concentrations(i.e. 1.0×10⁻⁸ M) of E₂ and E₁. In the present studies, the biphasiceffect of E₂ reported by Amara and Dannies (Amara J F and Dannies P S(1983) Endocrinology 112, 1141-1143) was not found. This may beexplained by the different conditions used to conduct the assays. Thematter of assay culture conditions with ER⁺ cells has been discussed(Zugmaier G et al. (1991) J Steroid Biochem Mol Biol 39, 681-685).Certainly however, the low E₂ concentration for ED₅₀ still speaks to aproblem with ERα as the mediating receptor. Furthermore, the patternreported in this Example is consistent with physiological facts. Tumorformation by GH cells was greater in W/Fu rats treated with 25 mgestrogen pellets than in untreated intact sexually mature females(Sorrentino J M et al. (1976) J Natl Cancer Inst 56, 1149-1154). Withouta doubt, supraphysiological levels of estrogens were most effective invivo. In contrast to estrogens, progesterone and cortisol had no effecton GH₄C₁ growth in culture FIG. 11. These steroids also did not promoteGH cell tumor growth in vivo (Sorrentino J M et al. (1976) J NatlCancer. Inst 56, 1149-1154). The findings with androgens and GH₄C₁ cellgrowth shown in FIG. 11 revealed another important contribution made bythe work in CDE serum supplemented cultures described herein. TheInventor had shown before that T promoted GH tumor growth in vivo(Sorrentino J M et al. (1976) J Natl Cancer Inst 56, 1149-1154). It wasproposed at that time that T was effective because it was metabolized toestrogens in the rat. Therefore, it was expected that T would beineffective in culture. The results in FIG. 11 confirm this expectation.In this case, the new culture methods permitted resolution of an issuearising from previous in vivo observations. The dose-response results inFIG. 11 fortify a conclusion arrived at earlier that cell culture can beused to uncover physiologically important new information not accessibleby in vitro methods (McKeehan W L et al. (1990) In Vitro Cell Dev Biol26, 9-23).

Dose-response Effects of Steroid Hormones with Hamster Kidney TumorCells in CDE Serum. To explore the utility of the new culture conditionsfurther, steroid hormone effects on the H301 Syrian hamster kidney tumorcells in D-MEM/F-12 containing 50% (v/v) CDE-horse serum wereinvestigated. This cell line has two unique characteristics. First,tumors form from H301 cells in Syrian hamsters only in response toexogenous estrogens (Sirbasku D A and Kirkland W L (1976) Endocrinology98, 1260-1272). It is very important to note that normal physiologiclevels in intact adult female hamsters do not support tumor formation(Sirbasku D A and Kirkland W L (1976) Endocrinology 98, 1260-1272). Itis thought that progesterone from the normal estrus cycle suppressesgrowth in response to physiological levels of estrogen (Kirkman H andRobbins M (1959) In: National Cancer Institute Monograph No. 1, NationalInstitutes of Health, Bethesda, Md.). Second, these cells only formtumors in response to estrogens. The other major classes of steroidhormones are ineffective in vivo. The relative effectiveness of thethree estrogens with H301 cells was investigated (FIG. 12). Theirpotency was E₂>E₁>E₃. As with rat tumor cells, E₃ was markedly lesseffective than E₂ or E₁. E₂ and E₁ required 1.0×10⁻¹¹ M and 1.0×10⁻¹⁰ M,respectively, to achieve significant (p<0.05) growth. The ED₅₀concentration of E₂ is about 5 to 9×10⁻¹¹ M. As expected from in vivoresults (Sirbasku D A and Kirkland W L (1976) Endocrinology 98,1260-1272), this concentration was higher than for the rat pituitarytumor cells (FIG. 11) or rat mammary tumor cells (FIG. 3). In fact, theywere as much as 100 to 1000-fold higher than for human breast cancercells (FIG. 10). In other tests shown in FIG. 12, progesterone,cortisol, T and DHT were all inactive. The higher estrogenconcentrations required for significant growth of the H301 cells inculture, coupled with the marked estrogen specificity as is seen in vivo(Sirbasku D A and Kirkland W L (1976) Endocrinology 98, 1260-1272),indicate that the medium conditions used in this study yieldedphysiologically germane results.

Dose-response Effects of Steroid Hormones with Human Prostatic CarcinomaCells in CDE Serum. In the final dose-response study, the potency ofseveral classes of steroid hormones with the LNCaP cells was analyzed.This was done in D-MEM/F-12 containing 50% (v/v) CDE horse serum. Due toa point mutation which permits binding of both androgen and non-androgenhormones to the AR of LNCaP cells (Veldscholte J et al. (1990) BiochemBiophys Res Commun 173, 534-540; Veldscholte J et al. (1990) BiochimBiophys Acta 1052, 187-194), the Inventor expected several classes ofsteroids to promote growth, albeit at concentrations compatible withtheir known affinities for the mutated receptor. This proved to be thecase, as shown in FIG. 13. DHT and E₂ were the most potent steroids. Infact, they were equipotent. Both caused significant (p<0.05) growth at1.0×10⁻¹² M. Contrary to other reports (Schuurmans A L et al. (1988) TheProstate 12, 55-64; Sonnenschein C et al. (1989) Cancer Res 49,3474-3481; de Launoit Y et al. (1991) Cancer Res 51, 5165-5170; Lee C etal. (1995) Endocrinology 136, 796-803; Kim I et al. (1996) Endocrinology137, 991-999), the present study did not find that high concentrationsof DHT inhibited LNCaP growth. The potency of the steroid hormonestested was DHT=E₂>T>E₁>progesterone>E₃>cortisol. As potencies declined,saturation densities also decreased. The observed relative steroidpotencies agreed with those of others (Bélanger C et al. (1990) Ann NYAcad Sci 595, 399-402), and correlated with the expected binding of thevarious classes of steroids to the mutated AR of the LNCaP line.Additionally, the presently disclosed methods offered the advantage ofgreater growth responses. The results in FIG. 13 not only lend supportto the view that cultures containing a high concentration of CDE serumyield physiologically relevant information, but they also demonstratethat the new charcoal extraction method disclosed herein effectivelydepletes several classes of steroid hormones.

Discussion of Example 4. The methods presented in this Example show thatmitogenic effects of estrogens and androgens can now be measured at thepicomoloar level. These highly sensitive assays can be usedadvantageously to assess clinical samples for inhibitor concentrations(after steroid depletion) of to establish that sufficient estrogens arepresent to cause growth possibly in postmenopausal women. Theconcentrations that are measurable fall well below radioimmunoassayconcentrations and will give an accurate measure of the active estrogen(i.e. unbound) versus the total determined by conventional proceduresakin to radioimmunoassays. The results provided herein present a newapproach to the question of why postmenopausal women have sufficientlevels of estrogens to promote breast cancer cell growth. It is wellknown that ≧65% of the breast cancers in postmenopausal women areestrogen receptor positive. The results herein indicate that thesecancers are so sensitive to estrogens that even a reduced physiologicalconcentration is sufficient to cause growth. Breast cancer prevention byanti-hormone therapy must be evaluated on this new basis.

The results demonstrate clearly that serum contains at least oneestrogen reversible inhibitor and that it/they mediates physiologicallyrelevant sex steroid responses. The fact that CDE-horse serum iseffective with several cell lines of rodent and human origins indicatesthat the inhibitor or inhibitors are not species specific. Moreover, thefact that all of the ER⁺ cell lines responded similarly in these studiesto the different types of serum tested indicates that the inhibitor orinhibitors are ubiquitous in mammals. This suggests an importantphysiologic fact. Estrogen target tissue growth is coordinate in vivo.Administration of the hormone causes mitogenic effects in all of themajor target tissues such as breast, uterus, ovaries, female genitaltract, pituitary and specialized other tissues and cells. Therefore, thestudies presented imply that the inhibitor or inhibitors should beactive with several target tissues.

The results presented in this Example have special significance withregard to support for the conclusion that a new ERγ regulates growth. Inthese studies, growth is one-half maximally stimulated by 10-1,800 foldlower concentrations of E₂ than indicated by the Kd values expected ofthe classical ERα. According to the accepted theory of hormone binding,the K_(d) value represents the steroid concentration that one-halfsaturates the existing receptors. The following Table 4 summarizes theED₅₀ concentrations required for a one-half maximum growth in mediumcontaining 30 to 50% (v/v) CDE-serum versus the estrogen receptor K_(d)measured for the same or closely related cell lines. The new receptor isdiscussed further in a later Example.

TABLE 4 Comparisons of ED₅₀ and K_(d) as Evidence Supporting a New ERDesignated ERγ Fold-higher K_(d) Concentration ED₅₀ for E₂ Compared toED₅₀ for Cell Line Induced Growth K_(d) for E₂ Growth MTW9/PL2 1 × 10⁻¹²M  1.8 × 10⁻⁹ M 1.8 × 10³ T47D 1 × 10⁻¹² M 0.11 × 10⁻⁹ M 1.1 × 10³ GH₄C₁1 × 10⁻¹¹ M 0.25 × 10⁻⁹ M 25 H301 9 × 10⁻¹¹ M 0.87 × 10⁻⁹ M 10

Example 5 Thyroid Hormone Growth Effects in CDE-Horse Serum Prepared at34° C.

In this Example an assay system is described for testing substancesexpected to have thyroid hormone like activity. GH rat pituitary tumorcells are highly thyroid hormone responsive in serum-free defined medium(Eby J E et al. (1992) Anal Biochem 203, 317-325; Eby J E et al. (1992)J Cell Physiol 156, 588-600; Sato H et al. (1991) In Vitro Cell Dev Biol27A, 599-602). An example of this responsiveness with the GH₃ line isshown in FIG. 14. However, in serum-free defined medium, these cells arenot E₂ responsive when T₃ is omitted from the medium (FIG. 15). Duringevaluation of the role the GH cell lines in CDE-serum, it was found thatin D-MEM/F-12 with 2.5% (v/v) CDE-horse serum, T₃ caused substantialgrowth of the GH₄C₁, GH₁ and GH₃ rat pituitary tumor cell lines (FIG.16). However, at 50% (v/v) CDE-horse serum, only supraphysiologicconcentrations of thyroid hormone showed growth effects (FIG. 17).Nonetheless, the 34° C. CDE method described in the preceding Examplesis clearly functional to demonstrate both steroid hormone and thyroidhormone growth effects in culture. It is known that the thyroid hormonereceptor is a member of a superfamily of receptors that also includesthe steroid hormone receptors (Evans R M (1988) Science (Wash DC)240:889-895). Testing of substances expected to have thyroid hormonelike activity can be performed with the GH cell lines in the presence oflow concentrations of CDE-serum.

Discussion of Example 5. The removal of thyroid hormones from serum hasbeen described before using the Bio-Rad™ AG-1 X8 ion exchange resin(Samuels B H et al. (1979) Endocrinology 105, 80-85). Removal of T₃/T₄by this method relies on their negative carboxylic acid charge atneutral pH. That method also removes most of the other lower molecularweight charged substances from serum. For some applications, this is notbeneficial, particularly to the demonstration of steroid hormoneresponsive cell growth in culture. Also, the ion exchange method doesnot remove the uncharged/hydrophobic steroid hormones. Therefore, theAG-1 X8 method is more limited than the 34° C. CDE method describedherein.

Example 6 Estrogenic Effects in XAD-4™ Resin Treated Horse Serum

Horse serum depleted of steroid hormones by XAD-4™, prepared asdescribed in Example 2, was assayed to determine if it demonstratedestrogen reversible inhibition of ER⁺ cancer cell growth in culture.FIG. 18 shows the effects of XAD-4™ treated horse serum±10 nM E₂ withthe MTW9/PL2 cell line. Unmistakably, the pattern of cell response wasthe same as seen with CDE-horse serum. At 50% XAD-4™ serum (v/v), anestrogenic effect of 5.2 CPD was observed in 7 days. FIG. 19 shows asimilar experiment with T47D cells after 14 days. At 50% (v/v) XAD-4™treated serum, an estrogenic effect with T47D cells of 5.3 CPD wasobserved. The magnitudes of the estrogenic effects with both cell lineswere the same as observed with CDE-horse serum. Because both MTW9/PL2and T47D cells are sensitive to picomolar concentrations of estrogen, itwas evident that the XAD-4™ resin treatment effectively removed theendogenous sex steroids present in serum.

Discussion of Example 6. There is no previous report of the preparationsteroid depleted serum by this resin treatment method. As indicated inExample 2, the XAD-4™ treatment method has particular applicability forthe industrial preparation of large volumes of steroid hormone depletedserum, and will allow the commercial supply of steroid depleted serum atreasonable cost. A preferred application for this steroid hormonestripped serum is in the biotechnology industry, in which cell cultureis used to produce medically and otherwise commercially significantproteins and cellular products. Steroid hormone depleted serum hasapplicability beyond the ER⁺ and AR⁺ cells described in this report. Forexample, hybridoma cells are the sources of many important monoclonalantibodies. Depletion of steroids from the serum used to grow thesecells will increase cell viability (e.g. cortisol is a potent cytotoxicagent for leukocyte cell types), and therefore increase product yield.Moreover, steroid-stripped sera prepared in this way may stabilizehybridoma production of desirable immunoglobulins. The use of XAD™-4extracted serum is also applicable to development of hybridoma cells ofmedical significance and therapeutic value. These and other applicationsof the XAD™-4 treated serum for both commercial and diagnostic testingas well as for industrial production of valuable cellular products areforeseen.

Example 7 Testing of Substances for Estrogenic Activity

The purported estrogenic effects of phenol red were tested and proven tobe unfounded. Further, the methods described in this Example exemplifymethods that are generally effective for assessing the steroidogenicactivity of any substance.

Examination of Phenol Red Indicator as an Estrogenic Substance. Thereported estrogenic action of phenol red and/or its lipophiliccontaminants has led to the widespread use of indicator free culturemedium to conduct endocrine studies in vitro (Berthois Y et al. (1986)Proc Natl Acad Sci USA 83, 2496-2500; Bindal R D et al. (1988) J SteroidBiochem 31, 287-293; Bindal R D and Katzenellenbogen J A (1988) J MedChem 31, 1978-1983). The generally accepted view is that the 8.1 mg/mL(i.e. about 23 μM) of phenol red present in the D-MEM/F-12 medium(Gibco-BRL) alone was sufficient to cause estrogenic effects. Despitethis, the results presented thus far in this disclosure show largemagnitude estrogen effects in D-MEM/F-12 tissue culture mediumcontaining the standard concentration of the indicator phenol red. Toensure that this potential problem was avoided in subsequent studies,the phenol red matter was further investigated, as reported(Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36,447-464). In so doing, nine estrogen receptor positive (ER⁺) cell linesrepresenting four target tissues and three species were selected. Phenolred was investigated using five different experimental protocols. First,E₂ responsive growth of all nine ER⁺ cells lines was compared in mediumwith and without the indicator. Second, using representative lines itwas determined whether phenol red was mitogenic in indicator freemedium. The dose-response effects of phenol red were compared directlyto those of E₂. Third, it investigated whether tamoxifen inhibitedgrowth equally in phenol red containing and indicator free medium. Thisstudy was based on a report indicating that antiestrogen effects shouldbe seen only in phenol red containing medium. Fourth, it wasinvestigated whether phenol red displaced the binding of ³H-E₂ using ER⁺intact human breast cancer cells. Fifth, it was investigated whether E₂and phenol red both acted as inducers of the progesterone receptor usinga human breast cancer cell line well known for this property (Horwitz KB and McGuire W L (1978) J Biol Chem 253, 2223-2228). All of theexperiments presented in this disclosure support the conclusion that theconcentration of phenol red contaminants in a standard culture mediumavailable today is not sufficient to cause estrogenic effects. Thestudies presented indicate that the real issue of how to demonstrateestrogenic effects in culture resides elsewhere than phenol red(Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36,447-464). It was found that demonstration of sex steroid hormonemitogenic effects in culture depends upon conditions that maximize theeffects of a serum-borne inhibitor(s). When the effects of the inhibitorare optimized, the presence or absence of phenol red makes no everydaydifference to the demonstration of estrogen mitogenic effects withseveral target cell types from diverse species (Moreno-Cuevas J E andSirbasku D A (2000) In Vitro Cell Dev Biol 36, 447-464).

Phenol Red Testing for Estrogenic Activity with MCF-7A Cells. Theoriginal reports of the effect of phenol red or its impurities had usedthe MCF-7 human breast cancer cells to assess estrogenic activity(Berthois Y et al. (1986) Proc Natl Acad Sci USA 83, 2496-2500; Bindal RD et al. (1988) J Steroid Biochem 31, 287-293; Bindal R D andKatzenellenbogen J A (1988) J Med Chem 31, 1978-1983). The initial studybegan with the MCF-7A strain of this population. As shown in FIG. 20A,growth was measured in the presence of increasing concentrations ofCDE-horse serum with and without phenol red in the medium and ±E₂.Concentrations of ≦10% (v/v) CDE-horse serum supported more than 5 CPD.Higher concentrations progressively inhibited in both indicatorcontaining and indicator free medium. In both types of medium, E₂ wasrequired to reverse the serum inhibition. To confirm that E₂ was equallyeffective in phenol red free and phenol red containing medium, theestrogenic effects shown in FIG. 20A were compared in both types ofmedium and at each serum concentration. The results of this analysis arepresented in FIG. 20B. The maximum estrogenic effect at 50% (v/v) serumwas 2.38 CPD (i.e. 2^(2.38) or 5.2-fold) in medium without indicator and2.56 CPD (i.e. 2^(2.56) or 5.9-fold) with phenol red. This differencewas not significant. Only at 5% (v/v) serum was there a significantly(p<0.05) greater estrogenic effect in phenol red free medium. However;in replicate experiments this<1.0 CPD effect was inconsistent. At allother serum concentrations, the growth differences between plus andminus phenol red were not significant.

Test of Phenol Red Effects with MCF-7K Cells. The MCF-7K strain wasroutinely more estrogen responsive than the MCF-7A line (Sirbasku D Aand Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446). TheMCF-7K cells also showed a serum concentration dependent growthinhibition (FIG. 20C). The final degree of inhibition at 50% (v/v) serumwas independent of phenol red. Only in the presence of 2.5, 5, 10 and20% (v/v) CDE-horse serum were the estrogenic effects significantlygreater in phenol red free (FIG. 20D). It is important to note thatwhile these differences were identified more often with the MCF-7Kstrain than the MCF-7A line, they were invariably small. Plainly, noserum concentration supported≧1.0 CPD estrogenic effects in phenol redfree medium compared to indicator free medium (FIG. 20D). In fact,deletion of phenol red improved estrogen responsiveness by an average ofonly 0.6 CPD with the MCF-7K line. When judged by the maximum estrogeniceffects achievable with MCF-7K cells in 50% (v/v) CDE-horse serum, plusand minus phenol red gave indistinguishable results of CPD 3.01(8.0-fold) and CPD 2.99 (7.9-fold), respectively (FIG. 20D).

Phenol Red Testing for Estrogenic Activity with T47D and ZR-75-1 Cells.The same experiments just described above with the MCF-7 cell strainswere repeated with T47D and ZR-75-1 cells. These lines weresubstantially more estrogen stimulated in CDE-serum than MCF-7 cells(Sirbasku D A and Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36,428-446) and hence were expected to be more sensitive to phenolred/contaminants.

Phenol Red and T47D Cells. T47D cells were grown in medium withCDE-horse serum both with and without phenol red (FIG. 21A). Lowconcentrations of serum (i.e. ≦2%) promoted growth. Higherconcentrations progressively inhibited growth irrespective of indicatorcontent. In both media, E₂ was required to reverse the inhibition (FIG.21A). In 50% (v/v) CDE-horse serum, the maximum E₂ responses were2^(5.35) (41-fold) and 2^(5.29) (39-fold) in phenol red containing andindicator free medium, respectively (FIG. 21B). Only at low serumconcentrations were phenol red effects observed in any experiment. Insome replicates, the phenol red effect was opposite to that expected.For example, in the experiment shown in FIG. 21B, 0.5 to 2.5% serumshowed significantly (p<0.05) greater estrogenic effects in the presenceof phenol red. These results graphically illustrate the hazards ofinterpreting 1.0 CPD responses either in favor of phenolred/contaminants as estrogens or in opposition to this proposal.

Phenol Red and ZR-75-1 Cells. ZR-75-1 cells showed similar results asthe T47D line. Serum caused an inhibition of growth that was undoubtedlyunrelated to phenol red (FIG. 21C). In both types of medium, and atevery serum concentration tested, E₂ was required to reverse theinhibition (FIG. 21C). In 50% (v/v) serum, ZR-75-1 cells showed maximumestrogenic effects of 2^(3.39) (10.5-fold) and 2^(3.49) (11.2-fold) inmedium with and without indicator, respectively (FIG. 21D). As seen withT47D cells, the ZR-75-1 line showed greater estrogenic effects in mediumwith phenol red than in medium without indicator when the serum was 0.5,5 or 10% (v/v) (FIG. 21D).

Phenol Red Testing for Estrogenic Activity with MTW9/PL2 Cells. The nextexperiments were done with MTW9/PL2 rat mammary tumor cells (FIG. 22A).They were inhibited by high concentrations of CDE-horse serum with andwithout indicator. E₂ was required to reverse the inhibition in bothtypes of medium (FIG. 22A). The maximum estrogenic effects in 50% serumwere 2^(5.82) (56-fold) and 2^(5.69) (52-fold) with and without phenolred, respectively (FIG. 22B). In the experiment shown in FIG. 22B,estrogenic effects were unpredictably greater in phenol red free mediumthan in medium with indicator. This was observed at low serumconcentrations (i.e. 0.5 and 1.0%) and again at higher levels (i.e. 20and 30%). Although suggesting a phenol red effect, these results in factonly serve to emphasize the pitfalls of accepting small changes asmeaningful even though they are significant at p<0.05. When estrogeniceffects were found with MTW9/PL2 cells in phenol red free conditions,they invariably were ≦1.0 CPD. The sum of the studies with MTW9/PL2cells did not yield a predictable correlation between estrogenic effectsin the absence of the indicator and serum concentrations.

Other Cell Lines Tested for Growth±Phenol Red and ±E₂. The resultspresented above were replicated with the GH₁ and GH₄C₁ rat pituitarytumor cell lines as well as with the H301 cells and the LNCaP cell line(Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36,447-464). Again, the presence or absence of the indicator in the mediumcontaining CDE-horse serum had no effect whatever on the demonstrationof the usual high estrogenic effects with these cells.

Direct Test of Phenol Red Estrogenic Activity. Three cell lines wereselected for a direct test of phenol red as a mitogen. The MCF-7A linewas used because it most closely approximated the origin and passage ageof the cells used to conduct the original study of phenol red as a weakestrogen (Berthois Y et al. (1986) Proc Natl Acad Sci USA 83,2496-2500). The T47D cells were chosen because they are the mostestrogen responsive human breast cancer cell line available today(Sirbasku D A and Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36,428-446). The MTW9/PL2 cells were chosen as an example of a highlyestrogen responsive rodent origin line (Moreno-Cuevas J E and Sirbasku DA (2000) In Vitro Cell Dev Biol 36, 410-427; Sirbasku D A andMoreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446). The assayswere done in phenol red free D-MEM/F-12 supplemented with 30% CDE-HS.This concentration was chosen even though it is not as inhibitory as 50%(v/v) serum. This selection was made to reduce possible interactions ofthe phenol red/contaminant with serum proteins while still retaining asignificant inhibitory effect. Phenol red concentrations of up to 16mg/L were added to this medium. This highest level was twice that instandard, commercially formulated Gibco-BRL D-MEM/F-12. Severaldifferent manufacturing lots of aqueous phenol red gave equivalentresults. The preparations used in this study ranged in age from newlyobtained to more than ten year old laboratory stocks. These experimentsgave unmistakable results. There was no increase in the growth of any ofthe cell lines in response to phenol red (FIG. 23A). By comparison,parallel cultures receiving E₂ showed sizable 2 to 5 CPD responses tothe natural hormone (FIG. 23B). E₂ at 1.0×10⁻¹⁰ M optimized growth ofall three cell lines. The ED₅₀ concentrations of E₂ were 3.0×10⁻¹² M.Significant (p<0.05) estrogenic effects were observed at 1.0×10⁻¹² M.The results presented in FIG. 23 indicate that the culture conditionsused in this study could reasonably be expected to detect responses dueto contaminants present at the concentrations indicated in the originalreports (Berthois Y et al. (1986) Proc Natl Acad Sci USA 83, 2496-2500;Bindal R D et al. (1988) J Steroid Biochem 31, 287-293; Bindal R D andKatzenellenbogen J A (1988) J Med Chem 31, 1978-1983).

Comparison of E₂ Potency in Medium with and without Phenol Red. Asdescribed above in Table 4, the T47D and MTW9/PL2 cells growsignificantly in response to 1.0×10⁻¹² M E₂. The D-MEM/F-12 used inthose studies also contained about 23 μM phenol red. When the results ofthose studies were compared to the experiments in FIG. 23B, done inD-MEM/F-12 without indicator, the estrogen dose response curves werevery similar. The conclusion is straightforward. E₂ dose-responses werenot affected by phenol red. If phenol red lipophilic contaminants werepresent at the concentrations originally suggested (Berthois Y et al.(1986) Proc Natl Acad Sci USA 83, 2496-2500; Bindal R D et al. (1988) JSteroid Biochem 31, 287-293; Bindal R D and Katzenellenbogen J A (1988)J Med Chem 31, 1978-1983) they should have masked the observation ofpicomolar effects of exogenous estrogens.

Effect of Phenol Red on Binding of ³H-E₂ to Intact Cells. For the nextstudy, intact T47D cells were used to measure the effects of phenol redon estrogen receptor binding. The cells were incubated with 5 nM ³H-E₂and the effects of addition of increasing concentrations of unlabeled E₂assessed (Table 5). A 100-fold excess of unlabeled E₂ displaced 75% ofthe binding of ³H-E₂. By this criterion, 75% of the binding of ³H-E₂ wasspecific to estrogen receptors (Chamness G C and McGuire W L (1975)Steroids 26, 538-542). The same analysis was conducted with aqueouspreparations of phenol red. Even at 16 mg/L, the indicator did notreduce the binding of ³H-E₂ (Table 5). This was true no matter whichbatch of indicator was analyzed (results not shown). The phenol red usedfor the experiment shown in Table 5 was approximately the same age(purchased in 1986) as the date of the original report (Berthois Y etal. (1986) Proc Natl Acad Sci USA 83, 2496-2500). These results raisethe question how often preparations of phenol red purchased at that timeas an aqueous membrane filtered product contained a sufficient level ofcontaminants to elicit an estrogenic effect.

TABLE 5 DISPLACEMENT OF ³H-E₂ BINDING TO INTACT T47D CELLS BY UNLABELEDE₂ OR UNLABELED PHENOL RED IN INDICATOR FREE AND SERUM-FREE D-MEM/F-12FOR TWO HOURS AT 37° C. Control - No Additions 12,458 ± 1615 100% (5 nM³H-E₂ only) 2.5 nM Unlabeled E₂ 12,177 ± 872 98% 5.0 nM Unlabeled E₂ 8,756 ± 588 70% 50 nM Unlabeled E₂  7,898 ± 744 63% 250 nM Unlabeled E₂ 4,892 ± 194 39% 500 nM Unlabeled E₂  3,494 ± 127 28% 1000 nM UnlabeledE₂  2,543 ± 304 20% 1 mg/L Phenol Red 12,670 ± 727 102% 2 mg/L PhenolRed 13,874 ± 906 111% 4 mg/L Phenol Red 11,730 ± 566 94% 8 mg/L PhenolRed 12,357 ± 664 99% 16 mg/L Phenol Red 13,748 ± 998 110%

Comparison of the E₂ and Phenol Red Induction of Progesterone Receptors.Another putative function of phenol red was to induce progesteronereceptors in estrogen sensitive cells. An investigation was made as towhether the indicator induced an increase in the progesterone receptorsof T47D cells which contain these sites (Horwitz K B et al. (1978)Cancer Res 38, 2434-2437). In a first study, the kinetics ofprogesterone receptor induction versus estrogen concentration in phenolred free medium were investigated (FIG. 24A). E₂ levels as low as1.0×10⁻¹² M caused a significant two-fold increase in receptor contentin four days. At 1.0×10⁻⁸ M, E₂ induced a four-fold increase inprogesterone receptors in four days. Clearly, E₂ induced a time andconcentration dependent increase in the progesterone receptors with T47Dcells. Next, this same analysis was done with phenol red over aconcentration range of 1 to 16 mg/L (FIG. 24B). Phenol red induced asmall increase in progesterone receptors at 8 and 16 mg/L after fourdays. This induction was about the same as caused by 1.0×10⁻¹⁴ M E₂(FIG. 24A). These results indicate that if estrogenic contaminants arepresent in phenol red, they are most likely in the 10⁻¹⁴ M range evenassuming equal receptor binding capacity to E₂. This point is importantbecause the active agent is thought to be only a trace impurity in manybatches of phenol red (Bindal R D et al. (1988) J Med Chem 31,1978-1983). The impurities bind to the estrogen receptor with only 50%of the affinity of E₂. The impurity was expected to be 0.002% of thephenol red concentration. Based on test results that employed manydifferent batches of Gibco-BRL D-MEM/F-12, this concentration of theimpurity seems highly unlikely in the medium commercially availabletoday.

Discussion of Example 7. The studies of the effects of phenol red or itslipophilic impurities demonstrate the usefulness of the presentlydisclosed methods for the assessment of estrogenic and androgenicactivity of commercially prepared materials, substances present orextracted from environmental or food sources or other sources that arethought to contain such activities. The testing can be approached bythree separate methods, as shown by examples with phenol red. (1)Compounds or other preparations and substances can be tested for growthactivity with human or rodent cell lines depending upon the informationsought. Potency can be established as UNITS based on E₂ or any otherestrogen or androgen required. This permits direct expression of theestrogen like activity or androgen like activity per volume or mass ofthe substance under evaluation. Levels can be measured without regardfor expensive development of a radio immunoassay that in the end stilldoes not yield evidence of biological activity as a sex steroid hormoneanalog (agonist or antagonist). The use of rodent cell lines opens thepossibility of direct comparison to in vivo activity if required. Theeffects of hormone-like substances can be tested with human cell linesin athymic nude mice or SCID mice as required. (2) Another form ofanalysis is direct measure of potency by ³H-E₂ or ³H-DHT bindingdisplacement analysis from whole cells or extracted estrogen receptors.An example with ³H-E₂ and whole cells is shown in Table 5. The twodifferent binding assays offer different information. Whole cells have apredominance of hydrophobic sites (i.e. membranes) that absorblipophilic substances and therefore may attenuate their activity. Use ofcell extracted sex steroid hormone receptors permits direct measure ofthe potential of a substance to act as a hormone independent of itsbiological effects. (3) Finally, use of the progesterone receptoranalysis permits evaluation of substances and preparations by a methodentirely independent of growth. This is a gene expression based analysisthat permits evaluation that can be used to supplement growth data or beused in place of growth analysis. The MTW9/PL2 cells have been shownabove to be suitable for this purpose.

Example 8 Testing of Substances for Inhibitor-like Activity

In studies described in this Example, TGFα, TGFβ1, EGF, IGF-I, IGF-IIand insulin were tested in the cell growth assay described in thepreceding Examples, substituting those proteins for the serum-borneinhibitor contained in the preferred CDE serum.

TGFβ1 as a Substitute for the Serum-borne Estrogen Reversible Inhibitor.Normal mouse mammary (Silberstein G B and Daniel C W (1987) Science(Wash DC) 237, 291-293; Silberstein G B et al. (1992) Dev Biol 152,354-362) and normal human breast epithelial cell growth is inhibited byTGFβ (Bronzert D A et al. (1990) Mol Endocrinol 4, 981-989).Additionally, human breast cancer cells are inhibited by TGFβ (Knabbe Cet al. (1987) Cell 48, 417-428; Arteaga C L et al. (1988) Cancer Res 48,3898-3904; Arteaga C L et al. (1990) Cell Growth Diff 1, 367-374). TGFβalso inhibits the GH₄C₁ rat pituitary tumor cells (Ramsdell J S (1991)Endocrinology 128, 1981-1990) and the LNCaP human prostatic carcinomacells (Schuurmans A L et al. (1988) The Prostate 12, 55-64; Wilding G etal. (1989) Mol Cell Endocrinol 62, 79-87; Carruba G et al (1994)Steroids 59, 412-420; Castagnetta L A and Carruba G (1995) Ciba FoundSymp 191, 269-286; Kim I Y et al. (1996) Endocrinology 137, 991-999). Instudies presented next, replacement of the serum-borne inhibitor withTGFβ was attempted. A number of related forms of this inhibitor areknown (Clark D A and Coker R (1998) Int J Biochem Cell Biol 30, 293-298;Massagué J (1998) Annu Rev Biochem 67, 753-791). TGFβ1 and TGFβ2 aremost often studied and commonly have similar potencies. For example,they are equipotent with human breast cancers cells (Zugmaier G et al.(1989) J Cell Physol 141, 353-361). TGFβ1 was chosen for the instantstudy. Without a doubt, a number of the key cell lines used throughoutthe Examples were inhibited by TGFβ. It was therefore consideredessential to ask if TGFβ was the estrogen reversible inhibitor.

TGFβ1 and MCF-7 Cells. Because MCF-7 cells are probably the most studiedhuman breast cancer line today, this next work began with those cells.TGFβ has been described as a hormone regulated autocrine inhibitor ofthe ER⁺ MCF-7 human breast cancer cell growth (Knabbe C et al. (1987)Cell 48, 417-428). In the present study, to test if TGFβ1 substitutedfor the serum-borne inhibitor with these cells, they were grown inD-MEM/F-12 containing 2.5% (v/v) CDE-horse serum plus increasingconcentrations of transforming growth factor and ±E₂. The results inFIG. 25A show that even 50 ng/mL of TGFβ1 caused only a modestinhibition of MCF-7K cell growth. Cell numbers were reduced from 350,000to 200,000 per dish. This difference was significant (p<0.05).Nevertheless, the estrogen reversal of the inhibition was no larger thanthe E₂ effect observed in D-MEM/F-12 containing 2.5% (v/v) horse serumwithout TGFβ1 FIG. 25A. Furthermore, when the cell number data wereexpressed as CPD (insert FIG. 25A), it was definite that TGFβ1 was atbest a very modest inhibitor and that there was no TGFβ1 relatedestrogenic effect.

TGFβ1 and MTW9/PL2 Cells. The next study was performed because theMTW9/PL2 cells are the only known estrogen growth responsive rat cellline derived from a hormone responsive carcinogen induced tumor. Asimilar analysis was done with the MTW9/PL2 rat mammary tumor cells(FIG. 25B). TGFβ1 reduced cell numbers from 350,000 to 100,000 per dish.This was significant (p<0.05). However, the presentation of cell numberresults only tends to exaggerate the effects of TGFβ1. When the resultswere converted to CPD (insert FIG. 25B), the actual inhibition was 1.5CPD. This was at most a 25% decrease in growth rate. As shown, there wasno estrogen reversal of the TGFβ1 inhibition with MTW9/PL2 cells.

TGFβ1 and other ER⁺ Cell Lines. The effects of TGFβ1 at 50 ng/mL±E₂ werealso investigated with the other cell lines used in this study. TheMCF-7A, T47D and ZR-75-1 human breast cancer cells were inhibited byTGFβ1 (FIG. 26A). From these results, and those in FIG. 25A, it wasclear that the MCF-7 cells were the most sensitive of the ER⁺ humanbreast cancer lines tested. Irrespective of the line, E₂ had noinfluence on the TGFβ1 mediated inhibitions (FIG. 26A). The sameexperiments were done with the LNCaP cells and the GH₄C₁ pituitary line(FIG. 26A). They were more sensitive to TGFβ1 than breast cancer cells.Nonetheless, the TGFβ1 effects were not reversed by E₂. When the cellnumber decreases presented in FIG. 26A were converted to CPD, it wasclear that the TGFβ1 effects were negligible and that E₂ was of nosignificant consequence (FIG. 26B). Thus, TGFβ1 did not substitute forthe estrogen reversible inhibitor(s) in CDE serum with any of the sexsteroid sensitive ER⁺ cell lines tested.

TGFα and EGF as Substitutes for the Estrogen Reversible Inhibitor in CDESerum. The EGF family of mitogens and receptors has been linked tobreast cancer proliferation, invasion and progression (Dickson R B andLippman M E (1987) Endocr Rev 8, 29-43; Norman no N et al. (1994) BreastCancer Res Treat 29, 11-27; Ethier S P (1995) J Natl Cancer Inst 87,964-973; de Jung J S et al. (1998) J Pathol 184, 44-52 and 53-57). Mostprominent among these polypeptide mitogens has been the EGF analogue,TGFα (Dickson R B and Lippman M E (1987) Endocr Rev 8, 29-43; de Jung JS et al. (1998) J Pathol 184, 44-52 and 53-57). Estrogen inducedsecretion of TGFα is thought to create an autocrine loop that promotesbreast cancer cell growth (Dickson R B et al. (1985) Endocrinology 118,138-142; Dickson R B et al. (1986) Cancer Res 46, 1707-1713; Dickson R Bet al. (1986) Science (Wash DC) 232, 1542-1543; Dickson R B and LippmanM E (1987) Endocr Rev 8, 29-43; Derrick R (1988) Cell 54, 593-595;Arrack B A et al. (1990) Cancer Res 50, 299-303; Kenney N J et al.(1993) J Cell Physiol 156, 497-514; Normanno N et al. (1994) BreastCancer Res Treat 29, 11-27; Dickson R B et al. (1987) Proc Natl Acad SciUSA 84, 837-841; Salomon D S et al. (1984) Cancer Res 44, 4069-4077; LiuS C et al. (1987) Mol Endocrinol 1, 683-692). TGFα is also thought topotentiate estrogen action in uterus (Nelson K G et al. (1992)Endocrinology 131, 1657-1664) as well as to regulate the EGF receptor inthis tissue (DiAugustine R P et al. (1988) Endocrinology 122, 2355-2363;Huet-Hudson Y M et al. (1990) Mol Endocrinol 4, 510-523; Mukku V R andStancel G M (1985) J Biol Chem 260, 9820-9824). The culture conditionsdescribed herein offer a new opportunity to test the autocrine growthmodel under conditions not previously available. Application of the newcell growth assays allowed a direct test to determine if anautocrine/intacrine growth factor loop explains the estrogen reversal ofthe serum inhibition.

EGF and TGFα as Substitutes for E₂. Growth of the MCF-7A, MCF-7K, T47Dand ZR-75-1 cells was measured in D-MEM/F-12 containing increasingconcentrations of CDE horse serum with and without exogenous EGF orTGFα. The results with the four cell lines are shown in FIGS. 27A, 27B,27C, and 27D, respectively. As expected, CDE horse serum wasprogressively inhibitory at concentrations>5% (v/v). The addition ofgrowth saturating concentrations (Karey K P and Sirbasku D A (1988)Cancer Res 48, 4083-4092) of EGF or TGFα did not reverse the effects ofthe serum-borne inhibitor. In control cultures without added polypeptidemitogens, E₂ completely reversed the serum inhibition. These resultsagain confirm the same conclusion arrived at earlier using an entirelydifferent approach (Karey K P and Sirbasku D A (1988) Cancer Res 48,4083-4092). Direct evidence for obligatory EGF/TGFα autocrine loops inestrogen responsive cell growth simply has not yet been established. Infact, there is solid in vivo evidence that challenges an EGF/TGFαautocrine loop as active in the action of estrogens (Arteaga C L et al.(1988) Mol Endocrinol 2, 1064-1069).

IGF-I, IGF-II and Insulin as Substitutes for Estrogen Action.Insulin-like growth factors I and II (IGF-I and IGF-II) promote breastcancer cell growth (Furlanetto R W and DiCarlo J N (1984) Cancer Res 44,2122-2128; Myal Y et al. (1984) Cancer Res 44, 5486-5490; Dickson R Band Lippman M E (1987) Endocr Rev 8, 29-43; Karey K P and Sirbasku D A(1988) Cancer Res 48, 4083-4092; Ogasawara M and Sirbasku D A (1988) InVitro Cell Dev Biol 24, 911-920; Stewart A J et al. (1990) J Biol Chem265, 2172-2178). IGF-I related proteins (Huff K K et al. (1986) CancerRes 46, 4613-4619; Huff K K et al. (1988) Mol Endocrinol 2, 200-208;Dickson R B and Lippman M E (1987) Endocr Rev 8, 29-43; Minute F et al.(1987) Mol Cell Endocrinol 54, 17-184, as well. IGF-II (Yee D et al.(1988) Cancer Res 48, 6691-6696; Osborne C K et al. (1989) MolEndocrinol 3, 1701-1709), are thought of as possible autocrine/paracrinemitogens. Their secretion in response to hormones has been proposed(Dickson R B and Lippman M E (1987) Endocr Rev 8, 29-43; Huff K K et al.(1988) Mol Endocrinol 2, 200-208; Osborne C K et al. (1989) MolEndocrinol 3, 1701-1709). Insulin itself is likely an endocrinemediator. In the instant study, it was investigated whether exogenousIGF-I addition to cultures containing CDE-horse serum substituted forthe inhibition reversing effects of estrogens with human breast cancercells. FIG. 28A and FIG. 28B show the results with the MCF-7K and MCF-7Acells, respectively. Clearly, 1.0 μg/mL IGF-I did not reverse the seruminhibition. This was true despite the fact that this concentration ofadded IGF-I was much more than growth saturating (Karey K P and SirbaskuD A (1988) Cancer Res 48, 4083-4092). Duplicate studies with the T47Dcells gave the same results (FIG. 28C). It should be noted that IGF-I isactive with breast cancer cells even in the presence of serum(Furlanetto R W and DiCarlo J N (1984) Cancer Res 44, 2122-2128; Myal Yet al. (1984) Cancer Res 44, 5486-5490; Osborne C K et al. (1989) MolEndocrinol 3, 1701-1709; Stewart A J et al. (1990) J Biol Chem 265,2172-2178; Cullen K J et al. (1990) Cancer Res 53, 48-53) that containsspecific growth factor binding proteins (Rechler M et al. (1980)Endocrinology 107, 1451-1459). Human breast cancer cells also secretebinding proteins for the insulin-like growth factors (Yee D et al.(1991) Breast Cancer Treat Res 18, 3-10). Binding of the insulin-likefactors to carrier proteins may attenuate activity (Zapf J et al. (1978)J Clin Invest 63, 1077-1084), have both inhibiting and activatingeffects (De Mellow J S et al. (1988) Biochem Biophys Res Commun 156,199-204), or enhance biological action (Elgin R et al. (1987) Proc NatlAcad Sci USA 84, 3254-3258; Blum W F et al. (1989) Endocrinology 125,766-772). In parallel studies (data not shown), the effects of IGF-IIwere assayed with the same breast cancer lines under the conditions usedwith IGF-I. Even at 500 ng/mL, IGF-II did not reverse the inhibitoryeffects of 10 to 50% (v/v) CDE serum. In another related test, insulinat 10 ng/mL to 10 μg/mL did not reverse the inhibition caused by 50%(v/v) CDE serum. The results with insulin, IGF-I and IGF-II weremutually supportive because these mitogens promote growth via a commonreceptor (Rechler M et al. (1980) Endocrinology 107, 1451-1459; Karey KP and Sirbasku D A (1988) Cancer Res 48, 4083-4092; Osborne C K et al.(1989) Mol Endocrinol 3, 1701-1709; Stewart A J et al. (1990) J BiolChem 265, 2172-2178). The insulin results were also important in anotherway. This hormone does not interact with binding proteins and hencetheir presence in medium will not influence insulin action. Theseresults again confirm the same conclusion arrived at earlier using anentirely different approach (Karey K P and Sirbasku D A (1988) CancerRes 48, 4083-4092). Direct evidence for obligatory IGF-1/IGF-IIautocrine loops in estrogen responsive cell growth simply has not beenconfirmed yet. In fact, there is solid in vivo evidence to the challengeIGF-1/IGF-II autocrine loop participation in the action of estrogens(Arteaga C L et al. (1989) J Clin Invest 84, 1418-1423).

Discussion of Example 8. From this series of experiments, it can bereadily appreciated that any other natural or synthetic protein or othersubstance can be similarly tested for cancer cell growth inhibitingactivity akin to the serum-derived inhibitor in the CDE horse serum.Also, the same XAD™-4 and CDE extraction protocols may also be appliedto body fluids and secretions other than serum, and the extracted fluidsmay be assayed as described for inhibitor activity. Such fluids orsecretions include plasma, urine, seminal fluid, milk, colostrum andmucus. An XAD™-4 column is especially suited for preparing a steroidhormone depleted specimen from a small sample of body fluid.

Conceptual Derivations from this Study. These results also have a directbearing on a number of hypotheses advanced to explain how estrogenscause target tissue cell growth. The development of the new methodsherein provided a unique opportunity to reevaluate the most widely citedproposals under consideration. It was concluded that serum contains aninhibitor that effectively blocks ER⁺ and AR⁺ cell growth. Furthermore,physiologic concentrations of sex steroid hormones reverse thisinhibition. The results were uniformly the same no matter from whichspecies the cell lines were derived or which species was the source ofthe serum. In every case, the effects of the various classes of steroidhormones on the different cell lines were consistent with their knowntumor forming/growth properties in vivo or published responses in vitro.These results provide new insights into the following proposedmechanisms.

Serum Factor Regulation—Demonstration of Estrogen Responsiveness. Theliterature describing positive sex steroid hormone growth effects isnotably weighted in favor of the use of serum-supplemented cultures. Infact, a review made of the literature (Briand P and Lykkesfeldt A E(1986) Anticancer Res 6, 85-90; Wiese T E et al. (1992) In Vitro CellDev Biol 28A, 595-602) indicates that most past studies have used mediumcontaining≦20% (v/v) steroid hormone depleted serum. Although otherinvestigators have reported estrogenic effects in “serum-free definedculture”, these studies actually used conditions that included aprolonged preincubation in the presence of serum (Allegra J C andLippman M E (1978) Cancer Res 38, 3823-3829; Briand P and Lykkesfeldt AE (1986) Anticancer Res 6, 85-90; Darbre P D et al. (1984) Cancer Res44, 2790-2793). The results presented in preceding Examples demonstrateclearly that large magnitude effects are readily demonstrable in mediumwith CDE-serum and that as the CDE-serum concentrations increase to amaximum useable level of 50%, cell growth is inhibited and estrogensinvariably reverse these effects. In light of those results, it wasclear that the presence of serum, or a factor(s) contained in serum,made possible the demonstration of sex hormone dependent growth inculture.

The Endocrine Estromedin Hypothesis—Positive Indirect Control. In 1978it was proposed (Sirbasku D A (1978) Proc Natl Acad Sci USA 75,3786-3790) that growth of estrogen target tissues was not mediateddirectly by these hormones, but was instead controlled indirectly bysteroid inducible circulating growth factors (i.e. endocrineestromedins). Estromedins were proposed to be secreted by target tissuessuch as uterus, kidney and pituitary, and to act in concert tosimultaneously promote the growth of all ER⁺ target tissues (Sirbasku DA (1978) Proc Natl Acad Sci USA 75, 3786-3790; Sirbasku D A (1981)Banbury Report 8, 425-443; Ikeda T et al. (1982) In Vitro 18, 961-979).The estromedin hypothesis arose from the observation that reproduciblein vitro direct estrogen mitogenic effects were not identifiable(Sirbasku D A (1978) Proc Natl Acad Sci USA 75, 3786-3790; Sirbasku D A(1981) Banbury Report 8, 425-443; Ikeda T et al. (1982) In Vitro 18,961-979). It must be emphasized that the original estromedin hypothesisrested entirely upon the failure to demonstrate large magnitude estrogenmitogenic effects in culture with cell lines confirmed to form steroidhormone responsive tumors in host animals. When estrogen effects wereclearly observed with the MTW9/PL2 rat mammary tumor cells in culture,as described herein and reported (Moreno-Cuevas J E and Sirbasku D A(2000) In Vitro Cell Dev Biol 36, 410-427; Sirbasku D A andMoreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446), it wasapparent that the endocrine estromedin model required furtherevaluation. It was reasoned that extension of these results toadditional ER⁺ cell lines, including those from other species anddiverse target tissues, would either provide important support for theearlier hypothesis or disprove it. In the work disclosed herein, thisreassessment has been accomplished. All of the ER⁺ cells tested, as wellas one androgen sensitive AR⁺ human cancer line, manifested substantialgrowth in response to the appropriate steroid hormones in culturescontaining inhibiting concentrations of CDE serum. There can be no doubtthat steroid hormones act positively to promote target tumor cellgrowth. The results presented in this report plainly nullify theprevious endocrine estromedin model of steroid hormone responsive cellgrowth. The disapproval of the earlier endocrine estromedin modelreopened the question of how estrogens and other factors regulate sexsteroid responsive growth.

The Autocrine and Paracrine Models—Positive Indirect Control. In thestudies described in this Example, it was investigated whether exogenousgrowth factors mimic the inhibitor reversing effects of estrogens. TheEGF/TGFα and insulin-like families were focused on because of their highbiological potencies and physiologic relevance. These growth factorswere expected to substitute for steroid hormones based on the autocrineloop mechanisms proposed earlier. Despite this expectation, polypeptidegrowth factors did not substitute for the estrogens. They were inactivein the presence of the serum-borne inhibitor. In point of fact,deduction indicates that it makes no practical difference whether thegrowth factors were autocrine or paracrine in origin. The presence ofthe serum inhibitor in effect blocks all mitogenic action except thatexerted by the steroid hormones. This is a preferred feature of theserum-borne inhibitor(s) disclosed herein, and is further described inExamples which follow, when the use of serum-free defined culture isdescribed. These results also indicate that the search for theregulatory mechanism controlling estrogen dependent growth must seek newdirections. Since the estrogenic effects seen in CDE-serum are thelargest yet recorded, CDE is the preferred source of the regulator inthe cell growth assays.

Culture Parallels in vivo Growth Regulation. The results shown in thisExample have another important implication. Usually normal in vivotissues are bathed in growth factor containing fluids. Mitogens withintissues may be of local origin or may be derived from the circulation(Gospodarowitz D and Moran J S (1976) Annu Rev Biochem 45, 531-558;Goustin A S et al. (1986) Cancer Res 46, 1015-1029). If growth factorshave unrestricted freedom to stimulate cell proliferation, normalformation and architecture of the tissues would not develop nor couldthey be maintained. Manifestly, tissue architecture would be disrupted.In fact, this is one definition of cancer (Sonnenschein C and Soto A M(2000)Molecular Carcinogenesis 29, 205-211). The properties of aserum-borne inhibitor that counterbalances unrestricted growth meritserious further consideration with regard to how cancers develop insteroid hormone sensitive tissues. Others researchers have also arrivedat this conclusion (Soto A M and Sonnenschein C (1985) J Steroid Biochem23, 87-94).

The Estrocolyone Hypothesis—Negative Indirect Regulation. Theestrocolyone model (Soto A M and Sonnenschein C (1987) Endocr Rev 8,44-52) is an indirect negative mechanism based on regulation of sexsteroid hormone dependent cells via a serum-borne inhibitor. Theinhibitor blocks growth promoted by non-steroidal mitogens such asgrowth factors and diferric transferrin. Sonnenschein and Soto firstproposed that estrocolyone acted at the cell surface via specificreceptors. The effects of sex steroid hormones were to bind estrocolyoneand prevent it from associating with the cells. Only low physiologicconcentrations of sex steroid hormones were needed for this function.The special emphasis of this model was that sex steroid hormones did notact through intracellular located DNA binding receptors (i.e. cytosolicor nuclear sites). These intracellular sites had no growth function.Hence, this was an indirect negative mechanism (Soto A M andSonnenschein C (1987) Endocr Rev 8, 44-52). The results presented inthis disclosure are in agreement with the serum-borne mediator aspect ofthe estrocolyone hypothesis. There is no doubt that serum from severalspecies contains a steroid hormone reversible inhibitor and that itsisolation and molecular characterization will be a major advance withboth practical and conceptual applications. With regard to the actionsite of the steroid hormones, these results differ from the estrocolyonehypothesis as described (Soto A M and Sonnenschein C (1987) Endocr Rev8, 44-52). The tentative identification of several estrocolyonecandidates have been described, and in U.S. Pat. Nos. 4,859,585(Sonnenschein) and 5,135,849 (Soto), the issue of properties was raisedagain, but with different conclusions than published earlier.

The Positive Direct Model—Steroid Hormone Receptor Mediation. The onemechanism most widely accepted regarding steroid hormones and growthinvolves the nuclear located DNA binding ERα receptor (Gorski J andHansen J C (1987) Steroids 49, 461-475). Growth is thought to bemediation by specific cytosolic and/or nuclear located receptors thatultimately alter DNA transcription to regulate gene activity. Resultsfrom many laboratories support this mechanism (Jensen E V and Jacobson HI (1962) Recent Prog Horm Res 18, 387-414; Gorski J et al. (1968) RecentProg Horm Res 24, 45-80; Jensen E V et al. (1968) Proc Natl Acad Sci USA59, 632-638; Jensen E V and DeSombre E R (1973) Science (Wash DC) 182,126-134; Anderson J N et al. (1974) Endocrinology 95, 174-178; O'MalleyB W and Means A R (1974) Science (Wash DC) 183, 610-620; Lippman M E(1977) Cancer Res 37, 1901-1907; Harris J and Gorski J (1978)Endocrinology 103, 240-245; Markaverich B M and Clark J H (1979)Endocrinology 105, 1458-1462; Katzenellenbogen B S (1980) Annu RevPhysiol 42, 17-35; Katzenellenbogen B S (1984) J Steroid Biochem 20,1033-1037; Clark J H and Markaverich B M (1983) Pharm Ther 21, 429-453;Darbre P et al. (1983) Cancer Res 43, 349-355; Darbre P D et al. (1984)Cancer Res 44, 2790-2793; Huseby R A et al. (1984) Cancer Res 44,2654-2659; Gorski J and Hansen J C (1987) Steroids 49, 46.1-475;Katzenellenbogen B S et al. (1987) Cancer Res 47, 4355-4360; O'Malley BW (1990) Mol Endocrinol 4, 363-369). As discussed elsewhere herein, thepreferred positive action of estrogens is activation of a new ERγ thatsaturates/activates at lower steroid concentrations than the ERα or theERβ.

Serum Proteins with Estrocolyone Steroid Binding Characteristics. If theestrocolyone mechanism is in fact correct, one must be able to identifyat least one serum protein with very high affinity binding (i.e. K_(d)picomolar) for sex steroids. There is, however, a major unresolvedproblem with that hypothesis. Other than sex hormone binding globulin(SHBG), additional high affinity estrogen binding in CDE human serum hasnot been found. SHBG has K_(d) of 1.7×10⁻⁹ M for E₂ at 37° C. (Rosner Wand Smith R N (1975) Biochemistry 14, 4813-4820). This affinity does notqualify as the high binding expected of estrocolyone. Also, a search forestrocolyone in human serum only resulted in identification of SHBG(Reny J-C and Soto A M (1992) J Clin Endocrinol Metab 68, 938-945). Nohigher affinity binding site/protein was found. The binding of labeledsteroid hormones with CDE-horse and CDE-rat serum was studied (resultspresented in an Example which follows), and ³H-E₂ specific binding atK_(d) of 20 to 50 nM was found. This is a significant matter becauseestrogenic effects are demonstrated in this disclosure at 1 to 10picomolar. As further support for this point, the estrocolyone authorsfound estrogenic effects at 10 to 30 picomolar E₂ (Soto A M andSonnenschein C (1985) J Steroid Biochem 23, 87-94; Soto A M andSonnenschein C (1987) Endocr Rev 8, 44-52). The lack of correlationbetween the concentration of steroid that promotes growth and affinityof sex steroids for serum components raises serious questions about thisaspect of the estrocolyone hypothesis. These observations also suggestthat a very high affinity intracellular ERγ regulates growth.

A New Model of Steroid Hormone Responsive Cell Growth. A new model bestfits the available data. It brings together aspects of both the directpositive mechanism and indirect negative control. According to thismodel, regulation of steroid hormone target tumor cell growth is abalance between positive and negative control signals. This balancedictates either growth (i.e. cell division) or quiescence (i.e. cellmetabolism and tissue specific function but without cell division).Direct positive control is mediated by a high sensitivity intracellularsex steroid receptor (yet to be defined) that ultimately activates geneexpression whereas negative regulation is exerted by a serum-borneinhibitor that acts at the cell surface. The results disclosed hereinsupport the view that growth is controlled directly by both negative andpositive mediators. The results presented further define the molecularproperties of the serum-borne inhibitor by eliminating TGFβ1 as acandidate. This is an important issue because of the well-known effectsof TGFβ on normal breast epithelial cells (Hosobuchi M and Stampfer M R(1989) In Vitro Cell Dev Biol 25, 705-713) and ER⁻ estrogen insensitivebreast cancer cells (Arteaga C L et al. (1988) Cancer Res 48,3898-3904). The results herein continue to confirm a previouslyunrecognized entity that serves as the estrogen reversible inhibitor inserum. Inhibitors that lack estrogen reversibility can be eliminatedfrom consideration.

Example 9 Serum-Free Defined Culture Medium Formulations

In this Example, formulations of various serum-free defined culturemedia are discussed. Among other features, the preferred embodiments ofthe present media provide useful tools for detecting estrogenic effects.

Serum-free Defined Mammalian Cell Culture—Development Background. Theuse of serum-free defined medium to grow diverse cell types in culturegained national and international recognition with the publication byHayashi and Sato (Hayashi I and Sato G H (1976) Nature (Lond) 259,132-134). They demonstrated a breakthrough. The serum supplementcommonly used in cell culture medium could be replaceable entirely bymixtures of nutrients and hormones in serum-free medium. Thisobservation was expanded to include cell types from many mammaliantissues (Barnes D and Sato G (1980) Anal Biochem 102, 255-270; Barnes Dand Sato G (1980) Cell 22, 649-655; Bottenstein J et al. (1979) MethodsEnzymol 58, 94-109; Rizzino A et al. (1979) Nutr Rev 37, 369-378).Further development and application of this technology has been reported(Barnes D W, Sirbasku D A and Sato G H (Volume Editors) (1984) CellCulture Methods for Molecular Biology and Cell Biology, Volume 1:Methods for Preparation of Media, Supplements, and Substrata forSerum-free Animal Cell Culture; Volume 2: Methods for Serum-free Cultureof Cells of the Endocrine System; Volume 3: Methods for Serum-freeCulture of Epithelial and Fibroblastic Cells; Volume 4: Methods forSerum-free Culture of Neuronal and Lymphoid Cells, Allan R. Liss/JohnWiley, New York). A national/international symposium organized anddirected by Drs. Gordon Sato, Arthur Pardee and David Sirbasku was heldat the Cold Spring Harbor Laboratory to address the unfolding technologyrequired for serum-free defined medium growth of cells in culture and todiscuss its applications (Sato G H, Pardee A B and Sirbasku D A (1982)Volume Editors, Cold Spring Harbor Conferences on Cell Proliferation,Volume 9, Books A and B, Growth of Cells in Hormonally Defined Media,Cold Spring Harbor, N.Y.).

Serum-free Defined Culture—Nutrient Additions. A number of nutrientadditions to D-MEM/F-12 are needed to grow the cells used in thepresently described studies. The formulations of serum-free definedmedium employed are specific optimizations, modifications, or necessarychanges of earlier media that have been described (Riss T L and SirbaskuD A (1987) Cancer Res 47, 3776-3782; Danielpour D et al. (1988) In VitroCell Dev Biol 24, 42-52; Ogasawara M and Sirbasku D A (1988) In VitroCell Dev Biol 24, 911-920; Karey K P and Sirbasku D A (1988) Cancer Res48, 4083-4092; Riss T L et al. (1988) In Vitro Cell Dev Biol 24,1099-1106; Riss T L et al. (1988) In Vitro Cell Dev Biol 25, 127-135;Riss T L and Sirbasku D A (1989) In Vitro Cell Dev Biol 25, 136-142;Riss T L et al. (1986) J Tissue Culture Methods 10, 133-150; Sirbasku DA et al. (1991) Mol Cell Endocrinol 77, C47-055; Sirbasku D A et al.(1991) Biochemistry 30, 295-304; Sirbasku D A et al. (1991) Biochemistry30, 7466-7477; Sato H et al. (1991) In Vitro Cell Dev Biol 27A, 599-602;Sirbasku D A et al. (1992) In Vitro Cell Dev Biol 28A, 67-71; Sato H etal. (1992) Mol Cell Endocrinol 83, 239-251; Eby J E et al. (1992) AnalBiochem 203, 317-325; Eby J E et al. (1993) J Cell Physiol 156, 588-600;Sirbasku D A and Moreno-Cuevas J E (2000) In vitro Cell Dev Biol 36,428-446).

Serum-free Defined Medium Nutrient Supplements—Bovine Serum Albumin.Bovine serum albumin (BSA) (Sigma Catalog No. A3912) was made by“initial fractionation by heat shock and Fraction V”, minimum purity 98%(electrophoresis), according to the supplier. A 50 mg/mL stock solutionof BSA was prepared in normal saline and was sterilized using 0.2 μmpore membrane filters. Aliquots are stored at −20° C. in plastic tubes.As will be discussed below, the “heat shock” step that was used in mostalbumin preparation methods inactivates the estrogen reversibleinhibitor disclosed herein.

Serum-free Defined Medium Nutrient Supplements—Linoleic Acid—Albumin(Lin-Alb). This preparation was purchased from Sigma as Linoleic AcidAlbumin Conjugate (Catalog No. L8384). The conjugate is supplied as apowder sterilized by irradiation. The fatty acid content is 1% (w/w)linoleic acid. A stock solution was typically prepared by dissolving thecontents of a 500 mg bottle in 10 mL of sterile normal saline to give afinal concentration of 50 mg/mL. Aliquots are stored at 4° C. inpolystyrene tubes. This solution is never frozen. Mammalian cells cannotproduce polyunsaturated fatty acids. They must be supplied in a solubleform. Fatty acids are carried physiologically bound to albumin.

Serum-free Defined Medium Nutrient Supplements—Ethanolamine (ETN). ETNwas purchased from Sigma (Catalog No. A5629) (FW 61). This liquid has adensity of 1.0117 grams/mL. Using 0.610 mL in 100 mL of water, a 100 mMstock solution was prepared which was sterilized using the 0.2 um poremembrane filters. The ETN was stored at −20° C. in polystyrene tubes.This nutrient is required to sustain phospholipid metabolism requiredfor all membrane biosynthesis.

Serum-free Defined Medium Nutrient Supplements—Phosphoethanolamine(PETN). This solid material was purchased as o-phosphoryl-ethanolamine(FW 141) (Sigma Catalog No. P0503). A 10 mM stock of PETN was preparedby dissolving 141 mg in 100 mL of water and sterilizing with 0.2 μm poremembrane filters. Aliquots were stored at −20° C. in polystyrene tubes.This component is an adjunct to ETN.

Serum-free Defined Medium Nutrient Supplements—Glutamine (GLUT). Thisessential amino acid was purchased from Sigma (Catalog No. G5763). It is“cell culture tested” according to the manufacturer. Addition ofglutamine (FW 146.1) to the culture media is necessary because of itsrelatively short half-life (i.e. about 80% is lost in 20 days at 35°C.). See the Sigma product information for the decay curves at differenttemperatures and pH. Purchased D-MEM/F-12 stored in the refrigerator forabout three weeks lost most of the original glutamine present. Forserum-free applications, additional supplementation is required tosustain growth. For a preparation, 11.7 g was dissolved in 400 mL ofwater to give 200 mM glutamine. This solution was sterilized using 0.2μm pore filter membranes. Aliquots are stored at −20° C. polystyrenetubes. The final glutamine concentration added to serum-free definedmedium is 2 mM. Glutamine is a major metabolite and energy source forcells growing in culture.

Serum-free Defined Medium Nutrient Supplements—Reduced Glutathione(GSH). Crystalline reduced glutathione (FW 307.3) was purchased fromSigma (Catalog No. G4251). A stock of 40 mg/mL was prepared bydissolving 400 mg in 10 mL of water. This stock was very quicklysterilized with a 0.2 μm pore filter unit. Aliquots were quickly storedat −20° C. in polystyrene tubes. According to Sigma technical service,this sulfhydryl (—SH) compound is unstable in aqueous solutions,including tissue culture medium, and is rapidly converted to theoxidized GS-SG form by exposure to air. Addition every two to four daysto the culture medium may be required for reducing agent requiringcells. Another reducing agent that also is effective is mercaptoethanol.It is more stable and often effective at lower concentrations than GSH.Preferably the concentrations are controlled effectively. Reducingagents act as “scavengers” of free radicals generated by the oxygenatmosphere of cell culture.

Serum-free Defined Medium Nutrient Supplements—Selenium (Se). A powderof sodium selenite (100 mg/vial) is obtained from Collaborative Researchor Sigma (Catalog No. S5261). It has been sterilized by irradiation. Thecontents of a single vial are dissolved in 100 mL of sterile water togive final stock of 1.0 mg/mL. This preparation should not be filtersterilized because Se binds to filters. The final volume was diluted to100 mL with sterile saline. Aliquots are stored at −20° C. inpolystyrene tubes. Selenium is an important cofactor for enzyme systemsthat protect the cells from oxidation effects.

Serum-free Defined Medium Nutrient Supplements—Diferric Transferrin(2FeTf). Iron Fe (III) saturated (98%) human transferrin (diferrictransferrin) was purchased from Collaborative Research (Catalog No.40304) or Sigma (Catalog No. T3309) as bottles containing 1 gram of redcolored powder. The contents of one bottle are dissolved in 100 mL ofnormal saline. This red colored solution is sterilized using 0.2 μM poremembrane filters. This stock is 10 mg/mL. Aliquots are stored at −20° C.in polystyrene tubes. All growing cells require diferric transferrin asa source of iron for a great many metabolic processes, except for a fewknown cell types in which free Fe (III) or chelated Fe (III) can besubstituted for diferric transferrin. The cell lines employed in thepresent Examples do not include those exceptional cell types, however.

Serum-free Defined Medium Growth Factor Supplements—Epidermal GrowthFactor (EGF). EGF prepared from mouse submaxillary gland (tissue culturegrade) was purchased from Collaborative Research (Catalog No. 40001) as100 μg in a sterile vial or from Sigma (Catalog No. E4127). The originalvials are stored at 4° C. according to the manufacturer's instructions.To prepare a stock solution, 5.0 mL of sterile saline was added to avial to yield a 20 μg/mL EGF solution. Aliquots are stored frozen at−20° C. polystyrene tubes. Repeated freeze-thaw must be avoided. Thisgrowth factor is useful because of its very broad cell specificityrange.

Serum-free Defined Medium Growth Factor Supplements—Acidic FibroblastGrowth Factor (aFGF). Acidic FGF is purchased from Sigma (Catalog No.F5542). It is the human recombinant product from E. coli. This producthas very specific handling requirements. It is provided sterilized in 25μg vials lyophilized from PBS containing 1.25 mg of BSA. The contents ofeach vial are reconstituted in 25 mL of sterile PBS containing 1.0 mg/mLof BSA and 10 μg/mL of heparin. Filtration of this product at thisconcentration must absolutely be avoided. This solution is stored at−20° C. in polystyrene tubes. The solutions of aFGF definitely cannot befreeze-thawed more than twice. This growth factor is highly labile.Careless handling will result in problems. Keratinocyte growth factor(KGF) can substitute for aFGF. The fibroblast growth factor family isimportant in growth of urogenitial tissues including bladder andprostate (Liu W et al. (2000) In Vitro Cell Dev Biol 36, 476-484).

Serum-free Defined Medium Growth Factor Supplements—Heparin. Heparin isused to stabilize FGF in cell culture (Gospodarowitz D and Cheng J(1986) J Cell Physiol 128, 475-484). Heparin is obtained from Sigma(Catalog No. H3149) as the sodium salt, Grade 1-A, from porcineintestinal mucosa. A solution of 1.0 mg/mL is made in saline andsterilized with 0.2 μm pore membrane filters. An aliquot of 250 μL isadded to the 25 mL of aFGF reconstitution solution used above. Sterileheparin is stored at 4° C.

Serum-free Defined Medium Adhesion Protein Supplement—Fibronectin (Fbn).Human plasma derived fibronectin can be purchased from many commercialsources. Bovine fibronectin is also available and is effective.Fibronectin is prepared from units of fresh human plasma (unfrozen) orfresh bovine (unfrozen) plasma by two methods (Retta S F et al. (1999)Methods in Molecular Biology 96, 119-124; Smith R L and Griffin C A(1985) Thrombosis Res 37, 91-101). Purity is evaluated by SDS-PAGE withCoomassie Brilliant Blue staining or silver staining (Pierce Chemicals®kits). Adhesion activity is confirmed with cells in serum-free definedmedium. Vitronectin can substitute for fibronectin.

Serum-free Defined Medium Iron (Fe (III) ChelatorSupplements—Deferoxamine mesylate (DFX). Deferoxamine (FW 656.8) ispurchased from Sigma (Catalog No. D9533). The stock solution is made at10 mM by adding 131 mg to 20 mL of highly purified water as describedabove. The solution is sterilized by filtration with 0.2 μM poremembranes. Aliquots are stored at −20° C. in polystyrene tubes.

Serum-free Defined Medium Iron (Fe (III) ChelatorSupplements—Apotransferrin (apoTf). Human serum apotransferrin can bepurchased from Sigma (Catalog No T4382). It is minimum 98% iron-free.Alternatively, apotransferrin is prepared, as described previously(Sirbasku D A et al. (1991) Biochemistry 30, 295-304; Sirbasku D A etal. (1991) Biochemistry 30, 7466-7477). Apotransferrin is prepared bydialysis against citrate buffer pH 5.0-5.5 with 1 μg/mL DFX present tochelate>98% of the iron. Handling and storage were as described fordiferric transferrin but with great care to avoid contact with ironsources.

Serum-free Defined Medium Nutrient Supplements—Bovine Insulin (INS).This hormone was purchased from either of two sources. From Gibco-BRL itis Insulin, Bovine Zinc Crystals for Cell Culture Applications (CatalogNo. 18125-039). It was also obtained from Collaborative Research(Catalog No. 40305) and stored at 4° C., according to thatmanufacturer's recommendation. Gibco-BRL recommends solid insulinstorage at −5° C. to 20° C. A stock of 10 mg/mL in 0.01 N HCl wasprepared by adding 250 mg of insulin to 25 mL of the acid. The HCl wasmade by adding 172 μL of concentrated (11.6 N) HCl to 100 mL of water.The final stock solution of 10 mg/mL of insulin is filter sterilizedusing 0.2 μm pore diameter membranes. Aliquots are stored at 4° C. inpolystyrene tubes. Care was taken not to freeze-thaw the aliquots ofstock solution. Insulin is a very broad range cell growth-stimulatingfactor as well as a regulator of specific metabolic processes. Atsufficiently high concentrations (i.e., usually >1 μg/mL, insulin causesgrowth via binding to the IGF-I Type I receptor (Karey K P and SirbaskuD A (1988) Cancer Res 48, 4083-4092).

Serum-free Defined Medium Nutrient Supplements—Thyroid Hormones. Thepreferred thyroid hormone is T₃ (3′, 5-Triiodothyronine (FW 673)),purchased from Sigma as Catalog No. T2752). It is stored desiccated at−20° C. To prepare stocks, 0.5 N NaOH was made by addition of 20 gramsof pellets to one liter of water. Then, 67.3 mg of T₃ was added. Afterdissolving the T₃ with stirring for a few minutes, 25 mL of this stockwas diluted up to 250 mL with water, for a final concentration of 0.05 NNaOH. This dilution was sterilized using the 0.2 μm pore diameterfilter. At this point, the final stock for storage was 10 μM T₃.Aliquots of this final stock are stored in polystyrene tubes at −20° C.The second thyroid hormone, thyroxin (T₄, sodium salt, pentahydrate FW888.9), is prepared by the same procedure. For this stock solution, 88.9mg of T₄ are used. T₄ is purchased from Sigma (Catalog No. T2501). T₄ isused at 10 to 20 times higher concentrations than T₃. Care is taken notto freeze-thaw these preparations. Thyroid hormones have a very broadrange of metabolic and growth effects, and many different types of cellsrequire thyroid hormones for growth in serum free culture.

Compositions of Serum-free Defined Media. TABLE 6 presents theformulations of the preferred serum-free defined media developed for usein detecting high-level steroid hormone reversible inhibition by steroidhormone-depleted (“steroid hormone stripped”) serum fractions and bypurified inhibitors in serum-free cell growth assays. As indicated inthe footnotes to the table, when a particular component is included inone of the formulations, the concentration that provides a suitable cellgrowth medium can fall within the indicated range.

TABLE 6 Composition of Serum-free Defined Media Based on StandardGibco-BRL D-MEM/F-12 CELL TYPE Human Human Rat Rat Hamster BreastProstate Mammary Pituitary Kidney MEDIUM NAME DDM-2MF CAPM DDM-2A PCM-9CAPM COMPONENT FINAL CONCENTRATIONS IN THE DEFINED MEDIA Insulin¹ 500ng/mL 10 μg/mL 10 μg/mL 10 μg/mL 10 μg/mL EGF² 20 ng/mL 20 ng/mL 20ng/mL None 20 ng/mL AFGF³ None 10 ng/mL None None 10 ng/mLTriiodothyronine⁴ 0.3 nM 1.0 nM 0.3 nM 1.0 nM 1.0 nM Diferrictransferrin⁵ 10 μg/mL 10 μg/mL 10 μg/mL 10 μg/mL 10 μg/mL Ethanolamine⁶50 μM 50 μM 50 μM 10 μM 50 μM Phosphoethanolamine⁷ 5 μM None 5 μM NoneNone Bovine Serum Albumin⁸ 500 μg/mL 1.0 mg/mL 500 μg/mL 500 μg/mL 1.0mg/mL Linoleic acid-BSA⁹ 150 μg/mL None 150 μg/mL None None Selenium¹⁰20 ng/mL 10 ng/mL 20 ng/mL 10 ng/mL 10 ng/mL Reduced glutathione¹¹ 20μg/mL None 20 μg/mL None None Glutamine¹² 2.0 mM None 2.0 mM None NoneHeparin¹³ None 7.5 μg/mL None None 7.5 μg/mL Deferoxamine¹⁴ 5 μM 10 μM 5μM 10 μM 10 μM Human Fibronectin¹⁵ 25 μg 20 μg None None 20 μg When acomponent is added, the following are the effective concentration rangesused: ¹INS range 100 ng/mL to 10 μg/mL ²EGF range 1 ng/mL to 50 ng/mL³aFGF range 0.2 ng/mL to 20 ng/mL ⁴T₃ range 0.3 nM to 10 nM ⁵2FeTf range2 μg/mL to 50 μg/mL ⁶ETN range 5 μM to 100 μM ⁷PETN range 5 μM to 50 μM⁸BSA range 0.2 mg/mL to 5.0 mg/mL ⁹Lin-Alb range 50 μg/mL to 500 μg/mL¹⁰Se range 5 ng/mL to 20 ng/mL ¹¹GSH range 1 μg/mL to 50 μg/mL ¹²Glutrange 0.5 mM to 2.0 mM ¹³Heparin range 1 μg/mL to 10 μg/mL ¹⁴DFX range 2μM to 20 μM ¹⁵Fbn range 15 μg to 50 μg per 35-mm diameter dish

Serum-free Media Variations. The variations described next areapplicable to the defined media in TABLE 6. Standard phenolred-containing Gibco-BRL D-MEM/F-12 is a preferred basal medium to whichthe defined media components are added. It contains 0.6 mM to 1.0 MCaCl₂. D-MEM/F-12 can be purchased from Gibco-BRL in the liquid form orcan be prepared from the powder formulation using only highly purifiedwater. Alternatively, another suitable basal medium could be used aslong as it provides at least the required minimum amounts of necessarynutrients, vitamins and minerals to maintain cell viability of thedesired cell line. The calcium concentration range preferred is 0.6 to10 mM. Calcium stabilizes the inhibitor in cell culture withoutimpairing cell growth. The human breast cancer cell medium, DDM-2MF, wasa modification of the original DDM-2 medium (Danielpour D et al. (1988)In Vitro Cell Dev Biol 24, 42-52) and MOM-1 (Ogasawara M and Sirbasku DA (1988) In Vitro Cell Dev Biol 24, 911-920) and contained modifiedhormone concentrations, deferoxamine (DFX) and fibronectin. Aqueous saltsolutions such as tissue culture medium contain hydrolytic polymericforms of Fe (III) (Spiro T G et al. (1966) J Am Chem Soc 88, 2721-2726).DFX binds this form of Fe (III) with very high affinity (Schubert J(1964) In; Iron Metabolism The Chemical Basis of Chelation, Springer,Berlin, pp 466-498). If not removed, Fe (III) inhibitshormone-responsive growth in serum-free defined medium (Sirbasku D A etal. (1991) Mol Cell Endocrinol 77, C47-C55; Sato H et al. (1992) MolCell Endocrinol 83, 239-251; Eby J E et al. (1993) J Cell Physiol 156,588-600; Eby J E et al. (1992) Anal Biochem 203, 317-325). The preferredcell growth media for conducting cell growth assays are substantiallydevoid of unbound Fe (III), i.e., preferably containing less than 1 μMFe (III), and more preferably containing no more than about 0.15 μM. Inpreferred growth assay systems described herein, which are substantiallydevoid of unbound Fe (III), the concentration of free, or active Fe(III) in the medium is less than a cell growth inhibiting amount.Fibronectin was used with DDM-2MF to promote cell attachment. The 35-mmdiameter assay dishes were pre-coated by incubation with the designatedamount of fibronectin (TABLE 6) for 16 to 48 hours at 37° C. in 2.0 mLof D-MEM/F-12. CAPM human prostatic cancer cell medium was developed tosupport the growth of tumor cells from this tissue. The composition ofCAPM is described in TABLE 6. CAPM also supports the growth of the H301Syrian hamster kidney tumor cells. DDM-2A, which is a modified form ofDDM-2 (Danielpour D et al. (1988) In Vitro Cell Dev Biol 24, 42-52), waspreferred for growing MTW9/PL2 cells. PCM-9 defined medium was developedfor growing the rat pituitary cell lines. This medium differs fromprevious PCM formulations (Sirbasku D A et al. (1991) Mol CellEndocrinol 77, C47-055; Sato H et al. (1992) Mol Cell Endocrinol 83,239-251; Eby J E et al. (1993) J Cell Physiol 156, 588-600; Eby J E etal. (1992) Anal Biochem 203, 317-325) in that DFX was substituted forapotransferrin and the triiodothyronine concentration was increased to1.0 nM. Although DFX and apotransferrin (2 to 50 μg/mL) are thepreferred chelators based on their very high specificity and affinitiesfor Fe (III), EDTA at 1 to 10 μM or sodium citrate at 10 to 1000 μM alsoeffectively neutralize the cytotoxic effects of Fe (III) (Eby J E et al.(1993) J Cell Physiol 156, 588-600). Ascorbic acid (vitamin C) alsochelates Fe (III), but is used less often because it is unstable in cellculture medium at 37° C. in an oxygen environment in the presence ofsalts and metals in the medium. Also, at concentrations of 50 to 100μg/mL, apo-ovotransferrin and apo-lactoferrin also were effective Fe(III) chelators in serum-free defined medium (Eby J E et al. (1993) JCell Physiol 156, 588-600). Although EGF, aFGF and insulin are thepreferred growth factors, several other human recombinant proteins areeffective. They have either been purchased or obtained as gifts fromGibco-BRL, Sigma or IMCERA Bioproducts. Insulin-like growth factors Iand II (IGF-I and IGF-II) can be used to replace insulin, transforminggrowth factor α (TGFα) replaces EGF, TGFβ as an inhibitory supplement,and basic fibroblast growth factor (bFGF) partially replaces aFGF.Insulin can be used to replaced IGF-I and IGF-II. All of these proteingrowth factors are dissolved under sterile conditions according tomanufacturers' instructions and stored as indicated.

Discussion of Example 9. The preferred serum-free media described aboveprovide an ideal scenario for the study of growth responses of hormoneresponsive cancers without the myriad of potential interactionsaccompanying the presence of serum with its 5000+ proteins and othercompounds. The formulations presented permit dissection of growth intoits individual parts caused by different stimulators. When of interest,a combination of a few factors can be investigated to achieve anunderstanding of growth promoter/inhibitor interactions (i.e.cross-talk). This is exceptionally difficult to achieve in the presenceof full serum. The serum-free medium described herein provided a toolfor the assessment of growth inhibitor(s) isolated from CDE-horse serum,whose actions are reversed by sex-steroid hormones, as mentioned at thebeginning of this Example and also discussed elsewhere herein. Theseserum-free defined media will allow direct analysis of the finalpurified serum-borne inhibitors under the most defined conditionsavailable for cell culture. This feature brings the regulation ofsteroid hormone dependence up to the conditions that have been the mostsought after over the past fifteen years. The preferred serum-free mediaof the present invention raise hope for the provision of new insightthat could help to clarify the mechanisms involved in the control ofbreast, prostatic and other mucosal cancers under conditions notpreviously available.

Moreover, because of widespread concern today about possiblecontamination of commercial animal sera by disease causing agents suchas bovine spongiform encephalopathy (“mad cow disease”), there is agreat need for serum-free cell culture media that can support a varietyof cell types. The new media compositions fill that need. The newserum-free media can be used not only for assays but also for largescale testing purposes and industrial uses such as cell cultureproduction of a desirable protein. For example, an antigen for vaccineproduction, or a monoclonal antibody can be prepared without fear ofcontamination by a serum-derived agent. The serum-free media are alsouseful for producing quantities of virus for vaccine manufacture or forproducing recombinant viruses for gene therapy, and can be substitutedfor a conventional serum-based medium in a basic cell culture method forproducing quantities of proteins or viruses. Such basic cell culturemethods are well known in the art and have been described in theliterature.

Example 10 Serum-Free Defined Medium Supports Both Hormone Sensitive andAutonomous Cancer Cell Growth

In this Example, it is shown that media derived according to the presentmethods are effective for supporting hormone sensitive and autonomouscancer cell growth.

Selection of Models to Study Hormone Dependence and Autonomy inSerum-free Defined Culture Media. One goal was to develop serum-freedefined media that can be used to directly compare negative serum factorregulation with steroid hormone responsive and steroid hormoneautonomous cancers of the same tissue. That meant establishing a mediumthat supported the growth of both cell types. As models, human prostaticcarcinoma and human breast carcinoma cells were chosen becauseresponsive and autonomous (unresponsive) cell lines are currentlyavailable for both types of cancers. Furthermore, as discussed above,these cancers have many common characteristics including their tendencyto pass from steroid hormone receptor positive to steroid hormonereceptor negative in a process called tumor progression. During thecourse of development of such defined media, one observation was madeconsistently: breast cancer cells that were ER⁺ (i.e. estrogensensitive) and prostate cancer cells that were AR⁺ (i.e. androgensensitive) grew less well in defined medium based on standard D-MEM/F12than in defined medium based on “low-Fe” D-MEM/F12. The results of anexample with T47D cells in DDM-2MF are shown in FIG. 29. The examplewith LNCaP cells in CAPM is shown in FIG. 30. Another example is thethyroid hormone responsive MDCK kidney tubule epithelial cells in CAPMas shown in FIG. 31. Standard D-MEM/F-12 contains both ferric nitrateand ferrous sulfate as nutrient additions. When purchased without thesesalts, the medium was designated “low-Fe” D-MEM/F-12. The ironconcentrations in standard and “low-Fe” D-MEM/F-12 were 1.0 μM and 0.15μM, respectively (Eby J E et al (1992) Anal Biochem 203, 317-325). Evenin “low-Fe” medium, iron is present as a contaminant in the chemicalsused to make the formulation, the 2.2 g/L NaHCO₃ added as a metabolicrequirement and buffer, and the 15 mM HEPES buffer necessary forstabilizing the pH under serum-free conditions (Eby J E et al (1992)Anal Biochem 203, 317-325). It is noteworthy that as low as 1.0 μM Fe(III) inhibits epithelial cell growth completely within five to sevendays. In another test the thyroid hormone responsive human HT-29 coloniccarcinoma cells in CAPM also grew better in “low-Fe” than standardD-MEM/F-12 (data not shown). This indicates that restriction of Fe (III)in culture medium will have implications even beyond sex steroid hormonedependent cells.

Modifications of the Usual Growth Assays for Experiments in “low-Fe”Medium versus “Standard” Medium. Specific modifications of the customarycell growth assays were required for assays done under iron-restrictedconditions. For example, the 35-mm assay dishes were incubated for 16 to24 hours prior with 20 to 25 μg of fibronectin in 2 mL of “low-Fe”D-MEM-F12 medium. Serum-free components were added to “low-Fe”D-MEM/F-12 at double the concentrations needed (2×) or to “standard”D-MEM/F-12 at (2×) as the experiments dictated. Each assay dish received1.0 mL of this solution. Next, the cells to be used in the assays werewashed three times in either “low-Fe” medium or “standard” mediumdepending upon the experimental protocol. These washes were done withthe same care as discussed above in the general materials and methodsdescribed in Example 1. Each dish received 1.0 mL of cells in theappropriate medium. At this point, the components final concentrationswere (1×) as summarized in TABLE 6. Also, TABLE 6 describes mediumcontaining deferoxamine as the Fe (III) chelator. Although lesspreferred, due in part to cost considerations, specificity, and affinityfor Fe (III), as noted above, apotransferrin is also effective,especially at the preferred apotransferrin concentration of 50 μg/mL.When apotransferrin binds Fe (III), it is converted to one of threeforms of ferric transferrin (Eby J E et al (1992) Anal Biochem 203,317-325). These three forms become additional support for cell growth indefined medium, thereby converting a toxic substance to a useablenatural nutrient.

Growth in Serum-free Defined Medium versus D-MEM/F-12 with 10% (v/v)Fetal Bovine Serum. To demonstrate the utility of the formulations inTABLE 6, cell growth was compared in serum-free defined medium±steroidhormone versus growth supported by fetal bovine serum. It is generallyaccepted that fetal bovine serum represents one of the most effectivesera for tissue culture. As an example, growth of the LNCaP cells wascompared in CAPM±DHT versus growth in 10% (v/v) fetal bovine serum (FIG.32). CAPM plus 10 nM DHT supported growth at about 80 to 90% of the rateof fetal bovine serum. Growth promoted by 10% fetal bovine serum,typically obtained from conventional commercial sources, reached 6.57(±0.48) CPD or, a 96-fold increase on cell number in 12 days. By day 12,cell densities in CAPM nearly equaled those in serum. Growth promoted bythe serum-free medium reached 6.22 (±0.35) CPD or 84-fold increase. CAPMwas able to support LNCaP growth even in the absence of sex-steroidhormones. Maximum growth obtained without sex-steroid hormones was of5.35 (±0.12) CPD or a 49-fold increase. The androgenic effect istherefore marginal, with differences of less than one CPD between thepresence and absence of DHT. Also shown, the cells did not grow inD-MEM/F-12 without any additions (FIG. 32). Similar studies were donewith other cell lines to determine growth rates versus serum and toestablish the periods for single time assays (e.g. 7, 10, 12 or 14days). FIG. 33 shows the same analysis with DU145 and PC3 cells in CAPMand in D-MEM/F-12 with 10% fetal bovine serum. As the cell number datashow, growth was logarithmic. After 12 days, growth in the serum-freemedium was identical to that in 10% fetal bovine serum for both celllines. Growth of PC3 in 10% serum reached 6.98 (±0.71) CPD or a 112-foldincrease in cell number versus 6.97 (±0.44) CPD or the same foldincrease for cell numbers in serum-free medium. Growth of DU145 in 10%fetal bovine serum was 6.71 (±0.58) CPD versus 6.73 (±0.18) CPD inserum-free conditions. The results in FIGS. 32 and 33 demonstrate byexample that the serum-free defined media in TABLE 6 are effective withboth hormone sensitive and hormone autonomous cells.

Determination of Component Concentrations and the Requirement for a Fe(III) Chelator. The optimum concentration of each single component wasdetermined by dose-response analysis in the presence of othercomponents. The technology used to establish early forms of serum-freedefined media has been described (Danielpour D et al. (1988) In VitroCell Dev Biol 24, 42-52; Ogasawara M and Sirbasku D A (1988) In VitroCell Dev Biol 24, 911-920). An example of this process is shown in FIG.34 with LNCaP cells. Dose-response effects of bovine serum albumin,apotransferrin, T₃, ethanolamine, selenium, and EGF are shown. Theresults show clearly that the addition of the iron chelatorapotransferrin was required for cell growth. After determining optimumconcentrations for each component, the contribution of each to the totalwas assessed by another assay. Individual components were deleted one ata time. As an example, the three most widely used prostatic carcinomacell lines were compared (i.e. LNCaP, PC3 and DU145) in CAPM thatcontained deferoxamine in place of apotransferrin (FIG. 35). Thedeletions were done±DHT. The first and most striking result was themajor differences between the growth requirements of the DHT sensitiveLNCaP cells and those of the autonomous DU145 and PC3. Only the deletionof diferric transferrin substantially prevented the growth of autonomouscells. Also, it was clear that deletion of deferoxamine had only a small(i.e. <20%) effect on growth of the DU145 and PC3 cells. The DU145 andPC3 cell lines also were T₃, insulin, EGF, fibronectin and deferoxamineindependent. As expected±DHT had no significant effect on DU145 or PC3.By contrast, LNCaP growth was significantly (p<0.01) reduced or arrestedcompletely by deletion of fibronectin, T3, diferric transferrin ordeferoxamine. LNCaP growth also was inhibited by deletion of EGF orinsulin, but these effects were pronounced only in the absence of DHT.

Discussion of Example 10. The media described in TABLE 6 were optimizedfor the specific cell types designated. Additionally, they wereoptimized to permit direct comparison of the growth properties of ER⁺and AR⁺ steroid hormone sensitive tumor cell lines to their ER⁻ and AR⁻steroid hormone insensitive (also called autonomous) counterparts. Thiscareful optimization was done originally to study rat mammary tumorcells of both types in DDM-2A defined media. The appropriate cell linesfor this approach have been developed from the MTW9/PL2 population anddescribed (Danielpour D and Sirbasku D A (1984) In Vitro 20, 975-980).The medium DDM-2MF has been developed for the same purpose only forcomparisons of ER⁺ and ER⁻ forms of these cancers. TABLE 1 lists themost important ER⁺ human breast cancer cell lines in use today. Inaddition a number of other ER⁻ human breast cancer cells lines have beenevaluated. They are the MDA-MB-231 (Cailleau R et al. (1974) J NatlCancer Inst 53, 661-674), BT-20 (Lasfargues E Y and Ozzello L (1958) JNatl Cancer Inst 21, 1131-1147), Hs0578T (Hackett A J et al. (1977) JNatl Cancer Inst 58, 1795-1806), MDA-MD-330 (Cailleau R et al. (1978) InVitro 14, 911-915), and the myoepithelial HBL-100 (Gaffney E V (1982)Cell Tissue Res 227, 563-568). The demonstration of ER status of theselines has been described (Reddel R R et al. (1985) Cancer Res 45,1525-1531). With regard to human prostatic cancer, the only reliableandrogen responsive cell line available today is the LNCaP (TABLE 1).Another, the ALVA-41, has been described as androgen growth responsive(Nakhla A M and Rosner W (1994) Steroids 59, 586-589). However, as shownin subsequent Examples, this line is autonomous by the criterion of alack of DHT effects in CDE-horse serum. Two other human prostate cancercell lines are commonly used as autonomous examples. These lines are theDU145 (Stone K R et al. (1978) Int J Cancer 21, 274-281) and the PC3(Kaighn M E et al. (1979) Invest Urol 17, 16-23). Previously, there wasa defined medium established for PC3 cells (Kaighn M E et al. (1981)Proc Natl Acad Sci USA 78, 5673-5676). This medium was evaluated and didnot support LNCaP cell growth. However, others have reported“serum-free” media that was stated to be effective with LNCaP, DU145,PC3 and ALVA-31 cells (Hedlund T E and Miller G J (1994) The Prostate24, 221-228). The problem was this medium was not serum-free nor was itdefined. The experiments began with cells plated into 5% serum and thenpreceded to use a serum fraction called fetuin to support growth. Fetuinis a complex undefined mixture of ≧4% of the proteins in serum. Underthose conditions, an accurate analysis of hormonal and growth factoreffects (Ogasawara M and Sirbasku D A (1988) In Vitro Cell Dev Biol 24,911-920) cannot be done satisfactorily. The completely serum-free CAPMin TABLE 6 supports the growth of all of these prostate cell lines. Inaddition, CAPM has been applied to the ER⁺ estrogen growth stimulatedH301 Syrian hamster kidney cells (Sirbasku D A and Moreno J E (2000) InVitro Cell Dev Biol 36, 428-446) and its autonomous derivative cell lineA195. As has been reviewed (Evans R M (1988) Science (Wash DC) 240,889-895), steroid hormones and thyroid hormones belong to the samesuperfamily of receptors. Both are important in growth. Therefore, itwas expected that some tissues might be thyroid hormone positiveregulated, while others might be positive regulated by steroid hormones.CAPM has also been applied to the study of thyroid hormone reversal ofpurified inhibitors with the human colon carcinoma cell line HT-29.Similar use has been made of CAPM with the MDCK dog kidney tubule cellline (Leighton J et al. Science (Wash DC) 158, 472-473). CAPM replaces adifferent defined medium prepared for MDCK cells (Taub M et al. (1979)Proc Natl Acad Sci USA 76, 3338-3342). It is likely that theprostaglandin in that earlier medium interfered with the action of thethyroid hormones. In any case, that medium was not useful fordemonstration of thyroid hormone reversal of purified MDCK cell growthinhibitors. All of these observations support the view that a series ofuniquely optimized media have been formulated to define the growthrequirements of epithelial cells from several of the very prominentcancers of humans. Furthermore, the technology developed promisesapplication to the optimization of growth of other types cells from avariety of epithelial/mucosal tissues. Epithelial/mucosal cancerscomprise 80% of those in humans.

Example 11 Differential Effects of Fe (III) on the Growth of HormoneResponsive and Autonomous Human Breast and Human Prostate Cancer Cells

This Example demonstrates that iron has an inhibiting effect on steroidresponsive cell growth, independent of the above-describedimmunoglobulin effects, and which is distinguishable from its effect onautonomous cells.

Approaches to Demonstration of Iron Toxicity. Standard D-MEM/F-12appeared to contain sufficient Fe (III) to inhibit hormone responsivecell growth (FIGS. 29 and 30). Accordingly, other approaches were usedto further demonstrate the deleterious effects of Fe (III) on hormoneresponsive tumor cell growth. To add Fe (III) to culture medium, it mustbe in a soluble form. Ferric ammonium citrate was selected for use.However, ferric ammonium sulfate is also effective. Other salts such asferric chloride or ferric nitrate or ferrous sulfate can be used. Ferricammonium citrate is a mixture that contains 16.6% of ferric iron byweight. The amount of mixture added to each dish was adjusted to achievethe desired Fe (III) concentrations. Due to the light sensitivity of themixture, the solutions were prepared fresh daily and the experimentscarried out under restricted light conditions. Also, the mixture wasprepared in water. Buffers without phosphate may be used, but they aregenerally less effective due to formation of insoluble materials. Theferric mixtures and the iron chelators EDTA, deferoxamine mesylate andsodium citrate were purchased from Sigma.

Iron Toxicity with Human ER⁺Breast Cancer Cells. In the firstexperiments, two ER⁺ cell lines were evaluated for Fe (III) sensitivityin DDM-2MF defined medium prepared with 10 μg/mL apotransferrin in placeof the deferoxamine shown in TABLE 6. The effect of addition of ferricammonium citrate on MCF-7A growth±E₂ at 10 days is shown in FIG. 36.Either with or without steroid hormone, Fe (III) was completelyinhibitory at 10 μM. There were no viable cells in the dishes at ≧10 μM.The EI₅₀ of Fe (III) with MCF-7A cells was 5 to 7 μM. A similar analysiswith T47D cells in DDM-2MF with 10 μg/mL apotransferrin instead ofdeferoxamine showed complete inhibition at 10 days with 2 μM Fe (III)(FIG. 37). At ≧2 μM there were no viable cells in the dishes either withor without (±) E₂. The EI₅₀ of Fe (III) with T47D cells was 1 μM.

Iron Toxicity with AR⁺ and AR⁻ Human Prostate Cancer Cell Lines. Theeffect of Fe (III) on AR⁺ LNCaP cell growth was assessed in CAPM definedmedium in which apotransferrin. (500 nM) was substituted fordeferoxamine, and the results are shown in FIG. 38. Clearly, 10 μM Fe(III) arrested growth to seed density levels (i.e. 12,000 cells perdish) in a 12-day assay. The EI₅₀ for LNCaP cells was 5 μM. In anotherexperiment in CAPM, the effects of ferric ammonium citrate wereevaluated with AR⁺ LNCaP cells and AR⁻ PC3 and DU145 cells (FIG. 39).Again, Fe (III) inhibited LNCaP cells to seed densities levels by 8 to10 μM. However, effects on the androgen autonomous PC3 and DU145 cellswere markedly less (FIG. 39). Reductions of 10 to 30% in cell'number forPC3 and DU145, respectively, were observed in 10 μM Fe (III). Theinhibitory effects of Fe (III) on the androgen independent PC3, DU145and ALVA-41 cells were variable, and never as marked as with the steroidhormone responsive LNCaP cells. The insert in FIG. 39 shows acorrelation between hormone responsiveness and Fe (III) effects. Theresults show a correlation between iron effects and thyroid hormoneresponsiveness. LNCaP cells are T₃ responsive whereas PC3 and DU145 arenot.

Reversal of Fe (III) Inhibition by Iron Chelators. Theinhibitory/cytotoxic effects of Fe (III) were reversible by the additionof iron chelators. Those studied were selected based on data showingtheir relative affinities and specificities for Fe (III) (Schubert J(1963) In: Iron Metabolism, Gross F, ed, Springer-Verlag, Berlin, pp466-496). Deferoxamine is most specific and has the highest affinity forFe (III). Citrate is next most effective. EDTA is not as effective noris it as specific as the first two chelators. In experiments with T47Dcells, the deferoxamine usually present in the DDM-2MF medium wasremoved and an additional 1.5 μM Fe (III) added to ensure completeinhibition of the cells. FIG. 40 shows the relative effects of additionof these three chelators to T47D serum-free defined medium cultures. Theorder of effectiveness was as expected from the affinities andspecificities of these chelators. Clearly, addition of Fe (III)chelators restored growth. FIG. 41 shows a similar study with LNCaPcells in CAPM defined medium from which the deferoxamine also wasremoved and 1.5 μM Fe (III) added. It was clear that chelation of the Fe(III) restored growth. It should be noted that this conclusion isreasonable based on the fact that deferoxamine has near absolutespecificity for Fe (III). Concentrations as low as 0.5 μM ofdeferoxamine were sufficient to induce 3.5 CPD with LNCaP cells. Maximumgrowth with this chelator (5.81 CPD) was obtained at 10 μM. Citrate andEDTA were also effective growth stimulators of LNCaP cells incubated athigh iron concentrations. Their maximum effects were with the additionof 500 μM and 10 μM respectively. The growth induction achieved withEDTA is lower than with citrate or deferoxamine. This probably could beexplained by the fact that EDTA is a less discriminatory chelator, andessential metals other than iron were affected. Concentrations of thechelators higher than those shown in FIGS. 40 and 41 were associatedwith cell damage and death. In particular, chelation of calcium bycitrate and EDTA will cause cell death in culture. The effect of thechelators was prevented by addition of more Fe (III) (data not shown).

Correlation Between Hormone Autonomy and Lack of Iron Effects. In thenext series of studies, data was sought supporting the concept that lossof steroid hormone dependence correlates positively with loss of Fe(III) effects. As shown in FIG. 30, LNCaP cells grew better in “low-Fe”serum-free defined medium than in defined medium based on “standard”D-MEM/F-12. This difference was also evaluated with the androgeninsensitive DU145 (FIG. 42) and PC3 (FIG. 43) cells. The results wereclear. The autonomous lines grew equally well in CAPM based on bothtypes of D-MEM/F-12. The presence of the higher Fe (III) level in CAPMbased on standard D-MEM/F-12 had no effect. To confirm that these celllines were androgen autonomous as defined by the loss of steroid andinhibitor growth regulation in CDE-serum, the next studies were done.DU145 cells showed no inhibition of growth in 50% CDE-serum (FIG. 44).There was no androgenic effect whatsoever. A similar assay with PC3cells showed essentially the same results (FIG. 45). There was noinhibition even in 50% CDE-horse serum, and no androgenic effect.Additionally, ALVA-41 cells are not iron sensitive (results not shown),and also are not sensitive to the serum-borne inhibitor (FIG. 46).

Discussion of Example 11. Together with the studies presented above, itappears that AR⁺ cells are sensitive to the serum-borne inhibitor,sensitive to the positive effects of steroid hormone and sensitive to Fe(III) inhibition. In contrast, the DU145 and PC3 cells are insensitiveto the serum-borne inhibitor, insensitive to the positive effects ofandrogen, and insensitive to Fe (R. The results presented in thisexample continue to demonstrate the requirement for the action of aserum-borne mediator to demonstrate steroid hormone responsive cellgrowth in culture. In addition, autonomy may be the loss of the receptorfor the serum factor and/or the loss of the intracellular steroidhormone receptor. If this hypothesis is correct it should be possible toidentify cells that possess steroid receptors but still have lost“sensitivity” to the hormone by virtue of the lack of the effect of theinhibitor. Most notably, this is the case with DU145 and ALVA-41 cells.As defined by immunohistochemistry, the DU145 cells are definitely AR⁺(Brolin J et al. (1992) The Prostate 20, 281-295). As defined by anumber of criteria, the ALVA-41 dells are AR⁺ (Nakhla A M and Rosner W(1994) Steroids 59, 586-589). A new concept explaining the progressionof normal tissue cells to hormone autonomous cancers is provided hereinand discussed in more detail in an Example below.

The use of CDE-serum is essential for the demonstration of androgen andother steroid hormone responsiveness in culture, but also limits theunderstanding of stimulatory or inhibitory roles of hormones or factorson prostate and other cancer cells because of the inclusion of anundetermined amount of undefined components. Serum-free medium willcircumvent this problem.

In these studies, it is clear that exposure of androgen responsiveprostate cancer cells to Fe (III) results in cell death. Compoundscontaining available Fe (III) offer the possibility of new therapies forprostate cancer localized to the tissue. It is proposed that deprivationof iron will be a highly effective means of eliminating the mostdangerous hormone autonomous forms of prostate cancer. The mostimpressive growth requirement of hormone autonomous prostate and breastcancer cells is for diferric transferrin as a source of essential ironfor growth. Without this iron source, none of the epithelial cancer cellexamined could proliferate. In fact, within a two to three week periodall cells in the cultures were dead.

The measurement of thyroid hormone receptors in prostate cancer shouldbe initiated as a diagnostic tool to determine iron sensitivity.Moveover, a new therapy mode for tumors containing mixtures of bothhormone responsive and autonomous cells is suggested, based on theobservation that deprivation of iron can equally kill both types ofcancer. This suggests that systemic Fe (III) therapy for disseminatedprostate cancer may be efficacious. It is definitely possible that ironin the Fe (III) form and compounds containing it will be effectiveanti-prostate cancer treatments, and that direct injection (or painting)of localized prostate tumors or metastasis at other sites (e.g. bone)might effectively kill these cancers without concomitant systemiceffects. This therapy potentially could replace such protocols assystemic chemotherapy (physically damaging), radiotherapy (damage tocollateral tissues) or the use of locally acting radioactive gold chipsthat are complex to handle in the surgical environment and must beimplanted and removed surgically. Furthermore, iron therapies can berepeated frequently by application via transrectal or transurethralaccess, using conventional techniques. This approach is unique and hasnot been discussed or suggested anywhere else in the literature. Suchiron treatments may be a useful therapy for benign prostatic hypertrophy(BPH). As discussed above, this condition is very common in older menand is treated usually by surgery. Application of iron compounds is anew approach to treatment of BPH. Iron treatment also offers a uniqueapproach to the problem of residual breast cancer cells in mastectomysites or after lumpectomy. The present studies suggest that these sitesbe “painted”, injected or otherwise treated locally with a Fe(III)-containing solution to destroy residual early (ER⁺) breast cancercells not detected at surgery. Subsequent treatments of these sites byinjection can be used as follow-up therapy alone or with the currentadjuvant chemotherapy or radiation therapy common in lumpectomy treatedpatients.

Example 12 Growth in Serum-Free Defined Medium Versus Growth inCDE-Serum±E₂

Use of Defined Media to Verify the Presence of a Serum-borne Inhibitor.The defined media described in Example 9 were used to verify thepresence of a serum-borne inhibitor. The growth of six different ER⁺cell lines was compared in serum-free defined media (TABLE 6) to theeffects seen in cultures supplemented with CDE-horse serum. Thesestudies are shown in FIGS. 47 and 48. Estrogenic effects are recordedfor each set of conditions with each cell line.

MCF-7K Cells in Serum-free and Serum Containing Medium±E₂. The firststudies were done with steroid hormone responsive human cancer celllines. FIG. 47A shows MCF-7K cell growth in serum-free DDM-2MF±10 nM E₂.The population replicated logarithmically for 12 days. E₂ had no effecton growth rate or saturation density. These results were in contrast toassays done in D-MEM/F-12 supplemented with CDE horse serum (FIG. 56B).Above 10% (v/v) serum, growth was progressively inhibited. Theinhibition caused by any serum concentration was reversed by E₂.Measured on assay day 10, a 3 CPD estrogenic effect was observed whichwas a 2³ or 8-fold cell number increase. The experiments were also donewith MCF-7A cells with similar results (data not shown). This effect inCDE-serum was as great as that reported for a special response clone ofthe MCF-7 cell line (Wiese T E et al. (1992) In Vitro Cell Dev Biol 28A,595-602).

T47D Cells in Serum-free and Serum Containing Medium±E₂. FIG. 47C showsthe growth of T47D cells in serum-free defined DDM-2MF±10 nM E₂.Although a small effect of estrogen was observed on growth rate, themost significant effect was an increase in stationary densities by 0.5to 1.0 CPD. In contrast, the effect of E₂ was much greater in mediumcontaining CDE horse serum (FIG. 47D). At 50% (v/v) CDE-serum, growthwas completely inhibited. The estrogenic effect under these conditionswas >5 CPD. This was more than a 2⁵ or 32-fold hormone effect on cellnumber. Comparison of these results with those of others (Chalbos D etal (1982) J Clin Endocrinol Metab 55, 276-283; Schatz R W et al. (1985)J Cell Physiol 124, 386-390); Soto A M et al. (1986) Cancer Res 46,2271-2275; Soto A M and Sonnenschein C (1987) Endocr Rev 8, 44-52; ReeseC C et al. (1988) Ann NY Acad Sci 538, 112-121) confirmed that theconditions in FIG. 47D were substantially more effective. Comparableexperiments with the ZR-75-I line gave results intermediate betweenMCF-7 and T47D cells (data not shown). ZR-75-1 cells showed no effect ofE₂ in serum-free defined DDM-2MF. This line grows more slowly than MCF-7or T47D cells in defined medium and in serum-supplemented cultures(Ogasawara M and Sirbasku D A (1988) In Vitro Cell Dev Biol 24,911-920). The maximum estrogenic effects of the preferred embodimentrecorded with ZR-75-1 cells in D-MEM/F-12 with 50% (v/v) CDE-horse serumranged between 3 and 4 CPD after 14 days. This was greater than reportedby others in serum containing (Darbre P et al. (1983) Cancer Res 43,349-355; Kenney N J et al. (1993) J Cell Physiol 156, 497-514) or“serum-free” medium (Allegra J C and Lippman M E (1978) Cancer Res 38,3823-3829; Darbre P D et al. (1984) Cancer Res 44, 2790-2793).

LNCaP Cells in Serum-free and Serum Containing Medium±E₂. In anotherstudy, the effects of E₂ on the growth of the LNCaP human prostaticcarcinoma cell lines in defined medium and in serum-supplemented culturewere compared. This cell line bears a point mutation in the AR thatpermits high affinity binding of estrogens to the altered receptor(Veldscholte J et al. (1990) Biochem Biophys Res Commun 173, 534-540;Veldscholte J et al. (1990) Biochim Biophys Acta 1052, 187-194). Inaddition, it is possible that estrogens cause LNCaP growth via aseparate functional ER (Castagnetta L A and Carruba G (1995) Ciba FoundSymp 191, 269-286). Irrespective of mechanism, estrogens are known topromote LNCaP growth (Bélanger C et al. (1990) Ann NY Acad Sci 595,399-402; Veldscholte J et al. (1990) Biochem Biophys Res Commun 173,534-540; Veldscholte J et al. (1990) Biochim Biophys Acta 1052, 187-194;Castagnetta L A and Carruba G (1995) Ciba Found Symp 191, 269-286). Aspresented herein (FIG. 47E), this cell line in serum-free defined CAPMshowed essentially no E₂ effect on growth rate and <1.0 CPD onsaturation density. When LNCaP growth assays were done in medium withCDE-horse serum, the mitogenic effect of E₂ was >5 CPD (FIG. 47F).Estrogenic effects herein were larger than reported by others with LNCaPcells in serum containing culture (Bélanger C et al. (1990) Ann NY AcadSci 595, 399-402; Castagnetta L A and Carruba G (1995) Ciba Found Symp191, 269-286).

LNCaP Cell Growth in CAPM Defined Medium with CDE-Horse Serum and ±DHTor E₂. To confirm that the serum-borne inhibitor can be assessed even inthe presence of all of the components of serum-free defined medium, anexample experiment is shown in FIG. 48. The LNCaP cells were grown inserum-free CAPM supplemented with increasing concentrations of CDE-horseserum without steroids and in assay dishes with the CDE-serum plus 10 nME₂ or 10 nM DHT. Without steroid, the CDE-horse serum showed theexpected progressive inhibition. Both the estrogen and androgen reversedthis inhibition completely at every serum concentration. Clearly, theinhibitor in serum possesses a very special quality that blocks theaction of the many mitogenic agents present in defined media.

GH₄C₁Cells in Serum-free and Serum Containing Medium±E₂. In the nextstudies, shown in FIG. 49, growth of rodent ER⁺ cell lines in definedmedium and CDE serum-containing medium with and without E₂ werecompared. The study was with the GH₄C₁ rat pituitary tumor cell line. Inserum-free PCM-9, E₂ had no effect on growth rate or saturation density(FIG. 49A). In contrast, the cells were highly estrogen responsive inCDE-horse serum (FIG. 49B). In 30% (v/v) CDE-serum, the estrogeniceffect was >4.5 CPD (i.e. >22-fold cell number increase). The GH₄C₁response obtained was substantially greater than that previouslyreported in cultures containing serum from a gelded horse (Amara J F andDannies P S (1983) Endocrinology 112, 1141-1143). Replicate studies withthe GH₁ and GH₃ rat pituitary tumor cells gave results equivalent tothose shown in FIGS. 49A and 49B (results not shown).

MTW9/PL2 Cells in Serum-free and Serum Containing Medium±E₂. FIG. 49Cshows the effect of E₂ on growth of the MTW9/PL2 rat mammary tumor cellsin serum-free DDM-2A. There was a small effect on growth rate and a 1.0CPD effect on saturation density. When the same cells were assayed inD-MEM/F-12 containing CDE horse serum, the effect of E₂ was remarkable(FIG. 49D). Cell number differences of 2⁶ (i.e. 64-fold) were recordedin 50% (v/v) serum in a seven-day assay. This result agrees with thosepresented above in this disclosure. Furthermore, comparison of MTW9/PL2responses (FIG. 49D) to those of the human breast cancer cell responses(FIGS. 47B and 47D) confirms that the ER⁺ rat cells are the mostestrogen responsive mammary origin line yet developed.

H301 Cells in Serum-free and Serum Containing Medium±E₂. In the finalstudies, the effect of E₂ on the growth of the H301 hamster kidney tumorcells in serum-free medium was compared to that in CDE horse serumcontaining medium. Estrogen had no effect on H301 cell growth inserum-free defined CAPM (FIG. 49E). In contrast, E₂ induced H301 cellnumber increases of >2⁴ (i.e. >16-fold) were recorded in D-MEM/F-12containing 30% (v/v) CDE serum (FIG. 49F). The H301 response was similarto the MCF-7 cells in that 50% (v/v) CDE-serum did not fully inhibit.The magnitude of the estrogenic effect with H301 cells was equal to thatreported by others studying this line in cultures supplemented with CDEserum prepared by different methods (Soto A M et al. (1988) Cancer Res48, 3676-3680).

Discussion of Example 12. The serum-free defined medium provide a modelsystem for identifying physiologically relevant new molecules. Whencompletely serum-free defined conditions were employed in the past, theeffects of estrogens were either marginal or insignificant as has beendiscussed above. The earlier observations in completely serum-freedefined culture medium have been extended in the present investigation.Direct comparisons were made between estrogenic effects in serum-freedefined culture and estrogenic effects in medium containing CDE serum.The results were unequivocal. With every cell line tested, CDE serum wasrequired to demonstrate significant estrogenic effects on logarithmiccell growth rates. A major advance provided was the clear demonstrationthat high concentrations of serum are required to observe largemagnitude estrogenic effects. Furthermore, the inhibitory effects ofserum are dose dependent even in the presence of the components used toformulate serum-free medium. This indicates that growth is progressivelynegatively regulated. This observation has physiological implications.Changes in the serum concentration of the inhibitor, or changes inavailability to target tissues, will have direct effects on the rate ofcell replication. The results in FIGS. 47 to 49 point to serum as thebest source yet identified to obtain the component that regulates sexsteroid responsive growth. The tissue origin of the serum regulatorremains to be investigated.

Example 13 Action of DES on Human AR⁺ LNCaP Prostate Cancer Cells

LNCaP Cells and DES Action. Diethylstilbestrol (DES) is now used as oneof the primary treatments for prostatic cancer (Seidenfeld J et al.(2000) Ann Intern Med 132, 566-577). Its action is likely mediatedthrough the hypothalamus-pituitary axis (Seidenfeld J et al. (2000) AnnIntern Med 132, 566-577). DES causes suppression of anterior pituitaryhormones (e.g. LH and FSH) and therefore suppresses testicular output ofandrogens. Although it is thought that DES has no direct effects onprostate cancer cells, the development of the assay methodology set outherein permitted a direct assessment of this issue. The AR⁺ LNCaP cellswere used as a model for these tests (FIG. 50). As shown in FIG. 50A, 10nM DHT effectively reversed the inhibition caused by higherconcentrations of CDE-horse serum in D-MEM/F-12. Likewise, 10 nM E₂ alsoreversed the CDE-serum caused inhibition completely (FIG. 50B). However,the same concentration of DES was entirely ineffective (FIG. 50C). DESdid not reverse the serum caused inhibition. The synthetic estrogen hadno direct positive effect on LNCaP cell growth. In the final study ofthis series, DES addition to medium containing DHT or E₂ did not affectthe reversal caused by these two natural steroids (FIG. 50D). Therefore,DES is not a direct inhibitor of androgen or estrogen promoted LNCaPcell growth. The view that DES acts indirectly to cause chemicalcastration is consistent with the present results. These results aresupported by other studies indicating that DES does not bind to the ARof LNCaP cells (Montgomery B T et al. (1992) The Prostate 21, 63-73).

Discussion of Example 13. The fact that DES is a major treatment forprostate cancer but does not act directly on the tissue has therapeuticimplications. For prostate cancer localized to the organ, or specificmetastases in other locations (e.g. bone, liver or lung), directapplication of Fe (III) offers a therapy with a different mode ofaction. It is also possible that local Fe (III) therapy (as described inExample 12) can be used in conjunction with conventional systemic DEStreatment to increase effectiveness above that with either treatmentalone. There is another potential advantage of local Fe (III) treatmentover systemic DES treatment. DES has many side-effects in males. Somepresent considerable discomfort or medical problems. Locally applied Fe(III) is absorbed by the body to form non-toxic mono ferric and diferrictransferrin by chelation with the large pool of availableapotransferrin. The iron containing proteins formed are no problem forthe body because they are the natural physiological forms of irondelivered to all tissues.

Example 14 Properties and Rationale for Serum Purification Source

Properties of the Serum-borne Inhibitor(s). It is clear from the resultspresented herein, and described in co-owned, concurrently filed U.S.Pat. No. ______ (Atty. Dkt. No. 1944-00201)/PCT/US2001/______ (Atty.Dkt. No. 1944-00202) entitled “Compositions and Methods forDemonstrating Secretory Immune System Regulation of Steroid HormoneResponsive Cancer Cell Growth,” which is hereby incorporated herein byreference, that charcoal-dextran treated serum contains a sex steroidhormone reversible inhibitor(s) of target tumor cell growth in culture.This activity was identified as a progressive cell growth inhibition inculture medium containing 10% to 50% (v/v) hormone depleted serum.Despite its first proposal more than fifteen years ago, until thepresent invention, the inhibitor had yet to be purified, partiallybecause of its instability. In an initial phase of investigations, ahighly enriched fraction of serum protein was produced whose estrogenreversible inhibitory activity was stable and whose cell growthinhibitory effects replicate those seen with full serum with a varietyof sex steroid hormone target tumor cell types in culture. Isolation wasfirst attempted using an array of standard protein purification methods.Although they were expected to enhance stability, inhibitor activity waseither not recovered after one only step or it was lost within twofractionation steps. In earlier work (Sirbasku D A et al. “Serum factorregulation of estrogen responsive mammary tumor cell growth.”Proceedings of the 1997 Meeting of the “Department of Defense BreastCancer Research Program: An Era of Hope”, (Abstract) pp. 739-740,Washington, D.C., Oct. 31-Nov. 4, 1997) indicated that the inhibitorshared some properties with sex hormone binding globulin (SHBG). Theseresults were obtained with a purification protocol known tosimultaneously yield purified corticosteroid binding globulin (CBG) andSHBG from human cord serum (Fernlund P and Lauren C-B (1981) J SteroidBiochem 14, 545-552). Additionally, it had been observed that the effectof calcium on both the estrogenic activity and the binding of ³H-DHT toCDE-serum was remarkably similar to data presented by others concerningthe stability of human SHBG (Rosner W et al. (1974) Biochim Biophys Acta351, 92-98). Different laboratories have raised the issue of classicalSHBG as the sex hormone reversible inhibitor of target cell growth. Thatprotein binds both androgens and estrogens in plasma and acts as acarrier system with cell signaling characteristics (Rosner W (1990)Endocr Rev 11, 80-91). However, in view of the results presented hereinand in U.S. Pat. No. ______ (Atty. Dkt. No.1944-00201)/PCT/US2001/______ (Atty. Dkt. No. 1944-00202), SHBG wasconsidered an unlikely candidate for the inhibitor. Both CDE-horse serumand CDE-rat serum contain concentrations of inhibitor about equal to anyof the other serum types investigated but they do not contain SHBG(Corvol P and Bardin C W (1973) Biol Reprod 8, 277-282; Renior J-M etal. (1980) Proc Natl Acad Sci USA 77, 4578-4582; Wenn R V et al. (1977)Endokrinologie 69, 151-156). Nevertheless, rabbit anti-human SHBGpurchased from Accurate Chemicals not only immunoprecipitated theestrogenic activity in CDE-horse and rat serum, but also precipitatedthe ³H-DHT (i.e. SHBG-like) binding activity in these sera. Thiscoincidence initially led to the mistaken conclusion that the inhibitorwas SHBG-like (Sirbasku D A et al. “Serum factor regulation of estrogenresponsive mammary tumor cell growth.” Proceedings of the 1997 Meetingof the “Department of Defense Breast Cancer Research Program: An Era ofHope”, (Abstract) pp. 739-740, Washington, D.C., Oct. 31-Nov. 4, 1997).This misconception turned out to be fortuitous, however, as it led to afurther exploration of the products obtained by the two-step cortisolagarose affinity and phenyl-Sepharose chromatography protocol. Thisprotocol, when used with horse and rat serum, provided material that atconcentrations of 10 to 15 μg/mL replicated the E₂ reversible inhibitioncaused by 30 to 50% (v/v) serum with steroid responsive human breastcancer cells, and responsive rat mammary, rat pituitary and Syrianhamster kidney tumor cells in culture. The inhibitor retained fullactivity for three years when stored unfrozen at −20° C. in the presenceof calcium, DHT and glycerol. As demonstrated herein, the long-standingproblem of inhibitor instability has been overcome, and a highly activepreparation became available to further probe molecular identity andmechanism(s) of action.

Mechanisms and Inhibitor Candidates. The regulation estrogen targettissue cell growth has been a topic of dynamic experimental interestbeginning several years ago (Jensen E V and DeSombre ER (1973) Science(Wash DC) 182, 126-134; O'Malley B W and Means A R (1974) Science (WashDC) 183, 610-620). Today, it is generally accepted that estrogeninteraction with specific nuclear located DNA binding receptors isnecessary to initiate critical cell cycle events (Dickson R B andStancel G M (1999) J Natl Cancer Inst Monograph No. 27, 135-145). It isalso highly likely that other non-steroid factors are essentialparticipants in this process (Sirbasku D A (1978) Proc Natl Acad Sci USA75, 3786-3790; Sirbasku D A (1981) Banbury Report 8, 425-443; Dickson RB and Lippman (1987) Endocr Rev 8, 29-43; Soto A M and Sonnenschein C(1987) Endocr Rev 8, 44-52). A number of years ago, studies werereported that indicated that serum-borne inhibitors, later named“estrocolyones”, had an important if not essential role (Soto A M andSonnenschein C (1985) J Steroid Biochem 23, 87-94; Soto A M andSonnenschein C (1987) Endocr Rev 8, 44-52). Estrocolyones were proposedto act as estrogen reversible inhibitors of steroid hormone targettissue cell growth. The results herein support this concept Over thecourse of several years, the inhibitor has been variously identified asan unstable M_(r) 70,000 to 80,000 protein (Soto A M et al. (1992) JSteroid Biochem Mol Biol 43, 703-712), the intact serum albumin molecule(Laursen I et al. (1990) Anticancer Res 10, 343-352; Sonnenschein C etal. (1996) J Steroid Biochem Mol Biol 59, 147-154), two domains of serumalbumin (Sonnenschein C et al. (1996) J Steroid Biochem Mol Biol 59,147-154) and SHBG (Reese C C et al. (1988) Ann NY Acad Sci 538,112-121). However, the roles of albumin and SHBG as estrogen relatedserum-borne growth regulators have been challenged (Sirbasku D A andMoreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 447-464;Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36,447-464; Soto A M et al. (1992) J Steroid Biochem Mol Biol 43, 703-712;Damassa D A et al. (1991) Endocrinology 129, 75-84). Prior to thepresent invention, no serum-derived inhibitor has been isolated, orotherwise identified at the molecular level, that replicates the largemagnitude estrogen reversible inhibitory effects of the presentlydisclosed inhibitors.

Discussion of Example 14. Purification of Source Serum. A goal of thesestudies was to obtain a high specific activity preparation of the seruminhibitor and to define isolation and storage conditions that willpermit its study over long experimental durations. Horse serum wasselected for the initial studies because it had several adventitiousproperties. First, it is a high content source of the estrogenreversible inhibitor that has biological activity with a broad range ofhuman and rodent sex steroid hormone target cells in culture. Second,when horse serum was steroid hormone depleted by charcoal extraction,the activity remained relatively stable at room temperature for a fewweeks. Third, horse serum did not contain SHBG. This bypassed the issueof classical M_(r) 94,000 dimeric SHBG as inhibitor. Additionally, horseserum is inexpensive, readily available, and presented minimum biohazardduring the application of the purification protocol.

Discovery Based on Serum Inhibitor Isolation. The fact that the estrogenreversible inhibitory activity was ubiquitous in mammalian serumsuggested that isolation from any one active species would lead toidentification in the others, possibly without purification. This isexactly what happened. The final estrogen-reversible inhibitors isolatedled to a major discovery of physiologic importance and revealed thefirst known link between the secretory immune system and mucosal cancerdevelopment and growth.

Example 15 Cortisol Affinity and Phenyl Sepharose Isolation of the“SHBG-Like” Estrogen Reversible Inhibitor from CDE-Horse Serum

Outcome of the Search for the Estrogen Reversible Inhibitors. As citedabove, neither horse or rat serum contains SHBG. Therefore, these werethe preferred sera to begin isolation. Partial purification of theinhibitor from serum has been achieved initially by a two-stepprocedure. The partially purified inhibitor fractions are different thanthe serum derived inhibitor described in U.S. Pat. No. 4,859,585 (issuedto Sonnenschein and Soto), which has been more recently identified as asubtype domain of albumin. By contrast, IgA and IgM, preferably indimeric/polymeric form, are steroid hormone reversible inhibitors ofcell growth. The discovery of immune regulation of sex hormone dependentgrowth is unique.

Two-step Cortisol-agarose and phenyl Sepharose Isolation Method. Basedon the perceived SHBG-like properties described above, a new approach tothe purification was taken. This method used a two-step cortisol-agaroseaffinity and phenyl-Sepharose chromatography protocol. It had beenemployed by others to simultaneously yield purified human cord serum CBGand SHBG (Fernlund P and Laurell C-B (1981) J Steroid Biochem 14,545-552). The method first required the synthesis of the cortisolaffinity matrix. The cortisol-agarose affinity matrix was synthesizedand the initial purifications done as described (Fernlund P and LarenC-B (1981) J Steroid Biochem 14, 545-552). An 80 mL bed volumecortisol-agarose column (2.5 cm×17.8 cm) was equilibrated with a buffercontaining 0.05 M piperazine, pH 5.5, with 0.2 M NaCl. Two liters ofhorse serum were charcoal-dextran extracted at 34° C. as describedabove. For two of the six preparations used in these studies, the serumwas depleted of steroid hormones by the Amberlite™ XAD-4™ resin method.There was no resulting difference in the purifications. After removing a30 mL sample for pre-column activity assay, the remaining volume wasadjusted to pH 5.5 with 1.0 N HCl. This was applied to the column at aflow rate of 30 to 40 mL per hour. Throughout the purification, the flowrates were maintained with a peristaltic pump. The effluent wascollected and a sample and adjusted to pH 7.2 for post-column assessmentof estrogen reversible inhibitory activity. After all of the serum hadbeen applied, the column was washed for 7 days at the same flow ratewith the equilibration buffer until the A_(280nm) of the effluent was<0.06 versus water.

To recover the activity, the cortisol-agarose column was eluted with a500 mL linear gradient formed with 250 mL of the piperazine/NaCl bufferand 250 mL of the buffer with 1.0 mg/mL cortisol and 10% (v/v) methanol.After completion of the gradient, the column was washed with one volumeof the cortisol/methanol buffer. A total volume of 600 mL was collectedas 10 mL fractions. As reported by Fernlund & Laurell (Fernlund P andLaurell C-B (1981) J Steroid Biochem 14, 545-552), two separateA_(280nm) or protein concentration ranges could be recognized, but theirseparation and individual chromatography on phenyl-Sepharose was no moreeffective than pooling the entire 600 mL gradient elution and using itfor the next step. The total volume from the cortisol gradient wasreduced 5 to 8-fold by nitrogen gas pressure Amicon ultrafiltration(YM-10 membrane) and applied directly to the next column withoutdialysis or pH adjustment.

A 28 mL bed volume phenyl-Sepharose (1.5 cm×16 cm) was equilibrated with0.05 M Tris-HCl, pH 7.5, containing 0.5 M NaCl. The concentratedcortisol gradient volume was applied at a flow rate of 60 mL/hour (10 mLfractions). The first A_(280nm) peak observed was a mixture of cortisoland CBG (Fernlund P and Laurell C-B (1981) J Steroid Biochem 14,545-552). These fractions were combined as cortisol affinity-phenylSepharose pool I (CA-PS-pool The column was then washed withequilibration buffer until the A_(280nm) was reduced to 0.002 versuswater. The next buffer applied was 0.05 M Tris-HCl, pH 7.5 (60%, v/v)containing 40% (v/v) ethylene glycol. The A_(280nm) peak observed withthis wash was combined to form CA-PS-pool II that corresponded to SHBGfrom human serum (Fernlund P and Laurell C-B (1981) J Steroid Biochem14, 545-552). The two pools were separately concentrated toapproximately 40 mL each and dialyzed separately against storage bufferwhich was 0.05 M Tris-HCl, pH 7.5, containing 0.15 NaCl, 0.05 M CaCl₂and 60% (v/v) glycerol. The dialysis further concentrated each sample.As last additions, 0.1 mM cortisol was added to CA-PS-pool I and 0.1 mMDHT was added to CS-PS-pool H. The pools were stored unfrozen at −20 C.Six replicate isolations were done. The protein yields ranged from 22.8to 37.7 for CA-PS-pool I and 5.82 to 12.2 mg for CA-PS-pool II. Based onan average of 60 grams of protein per two liters of CDE-horse serum(i.e. 30 mg/mL), CA-PS-pool II represented about 0.013% of the totalprotein in serum.

Cortisol affinity and phenyl Sepharose Isolation Results and SDS-PAGEMolecular Weight Estimation. The chromatography profiles from thetwo-step cortisol affinity and phenyl Sepharose isolation of theinhibitor(s) activity from CDE-horse serum are shown in FIG. 51. Theelution from phenyl Sepharose gave the CA-PS-pools I and II. CA-PS-poolI contained predominantly 58 kDa CBG (Rosner W and Bradlow H L (1971) JClin Endocrinol Metab 33, 193-198) as confirmed by SDS-PAGE and Westernimmunoblotting with rabbit anti-horse CBG as well as by partial aminoacid sequencing of the first 10 to 20 residues (results not presented).SDS-PAGE analyses of three example preparations of CA-PS-pool II areshown in FIG. 52A. Components of 67, 58, 54, and 29 kDa were identified.These were compared to the 48 and 46 kDa units identified for purifiedhuman SHBG (Khan M S et al. (1985) Steroids 45, 463-472) (FIG. 52A).

Native Molecular Weight Estimation. Analyzes done under non-reducing andnon-denaturing conditions using Superdex molecular sieve FPLC at neutralpH in buffers identified components CA-PS-pool I in the exclusion volumeat ≧900 kDa, and components approximately 350 and 168 kDa (Sirbasku D Aet al. “Serum factor regulation of estrogen responsive mammary tumorcell growth.” Proceedings of the 1997 Meeting of the “Department ofDefense Breast Cancer Research Program: An Era of Hope”, (Abstract) pp.739-740, Washington, D.C., Oct. 31-Nov. 4, 1997). Comparison of theresults from denaturing and non-denaturing conditions confirmed that theCA-PS-pool II was still heterogeneous and that the activity was mostlikely a subunit containing high molecular weight protein(s).

Removal of Storage Solution Components before Bioassay. Beforeconducting bioassays of the inhibitory activity in the phenyl-Sepharosepools, the glycerol and steroid hormones in the storage buffers wereremoved. If DHT is not removed completely from CA-PS-pool II, theinhibitory activity was substantially diminished or eliminated entirely.Samples (0.5 to 15 mL) were introduced into Slide-A-Lyzer® (Pierce)cassettes of molecular weight cutoff 10,000. The cassettes were incubatetwice with stirring in two liters of Tris-HCl, pH 7.4, containing 10 mMCaCl₂ for four hours at 34° C. to remove excess free steroids andglycerol. Next, the cassettes were transferred to the same buffercontaining 20% (v/v) of a charcoal-dextran mixture prepared as describedabove. After 18 hours at 37° C., the cassettes were transferred toanother two-liter volume of the same buffer containing 10% (v/v) of thecharcoal-dextran mixture and dialysis continued with stirring foranother 6 to 8 hours. Finally, the cassettes were rinsed lightly withwater and the dialyzed material recovered according to manufacturersinstructions. The contents were sterilized by 0.2-μm-pore membranefiltration and stored at 4° C. These preparations were usually usedwithin a few weeks.

Assay of CA-PS-pool I Estrogen Reversible Inhibitory Activity withMTW9/PL2 Cells. When assayed with MTW9/PL2 cells, CA-PS-pool I contained20 to 25% of the units of estrogen reversible inhibitory activityrecovered from the phenyl Sepharose column (data not shown). With twopreparations not presented, the cortisol gradient pool shown in FIG. 51was made 1.5 M NaCl before application to the phenyl Sepharose columnequilibrated at the same higher salt concentration. Under theseconditions, the CA-PS-pool I contained>90% CBG, as estimated bySDS-PAGE, but showed either no estrogen reversible activity or onlytraces (results not presented). Irrespective of the ionic strength or pHof the cortisol affinity pool applied to phenyl Sepharose, ethyleneglycol was required to elute the majority of the activity.

Assay of CA-PS-pool II Estrogen Reversible Inhibitory Activity withSeveral ER⁺ Cell Lines. Despite method variations with phenyl Sepharose,CA-PS-pool II always contained≧75% of the activity recovered. In acrucial test of significance, CA-PS-pool II was assayed to determine ifit replaced the effects of CDE-serum with eight different ER⁺ celllines. The results are shown in FIG. 53. The estrogen reversibleinhibitory effects of CA-PS-pool II were investigated with five rodenttumor cell lines derived from three different estrogen target tissuetumors, and three separate estrogen sensitive human breast cancer celllines. The cells were added to medium with 2.5% (v/v) CDE-horse serumplus increasing concentrations of CA-PS-pool II±10 nM E₂. The firstlines evaluated were the GH₁, GH₃, and GH₄C₁ rat pituitary tumor cells(FIGS. 53A, 53B and 53C, respectively). They were chosen first becausethese lines are well known for hormone regulation of differentiatedtissue specific functions in culture and exceptional sensitivity to avariety of hormones including estrogens (Tashjian A H Jr (1979) MethodsEnzymol 58, 527-535; Haug E and Gautvik K M (1976) Endocrinology 99,1482-1489; Haug E (1979) Endocrinology 104, 429-437; Amara J F andDannies P S (1983) Endocrinology 112, 1141-1143). At 10 μg/mL,CA-PS-pool II was fully inhibitory with all three GH lines. Growth wasreduced to near seed density levels (i.e. <0.5 CPD). By thismeasure, >1,700-fold increase in potency had been achieved versus fullCDE-serum. The ED₅₀ with the GH cells was 6 to 8 μg/mL which was a 300to 800-fold specific activity increase compared to full serum. E₂reversed the effects of the CA-PS-pool II at every inhibitoryconcentration. CA-PS-pool II replaced the effects of full CDE-serum withthese cells. FIGS. 53D and 53E show similar experiments with theestrogen sensitive H301 hamster kidney tumor cells and the MTW9/PL2 ratmammary cells, respectively. CA-PS-pool II was most inhibitory at 15μg/mL with both lines. The ED₅₀ were in the range of 5 to 10 μg/mL. Aswith the GH lines, E₂ completely reversed the effects of the inhibitor.Again, CA-PS-pool II replaced the effects of full CDE-serum with thesecells. With human breast cancer cell lines MCF-7K, ZR-75-1 and T47D, theresults were similar (FIGS. 53F, 53G, and 53H, respectively). Additionof 10 to 15 μg/mL of CA-PS-pool II caused maximum inhibition. The ED₅₀concentrations were 6 to 9 μg/mL. As with ER⁺ rodent cell lines, E₂completely reversed the inhibition caused by CA-PS-pool II. Again,CA-PS-pool II replaced the effects of full CDE-serum with these cells.

Cortisol-agarose Affinity Removal of the Inhibitor from CDE-serum. Nextit was determined if the cortisol affinity chromatography had notremoved the majority of the activity from serum. To test this, threecell lines were analyzed with pre- and post cortisol column samples.FIGS. 54A and 54B show the effect of a single column passage on theinhibitory activity for T47D human breast cells. The ED₅₀ of thepre-column CDE-serum was 7% (v/v) (FIG. 54A). Post-column, even 50%(v/v) serum did not achieve ED₅₀ (FIG. 54B). FIGS. 54C and 54D show thesame studies with the GH₃ rat pituitary cells. In this case, a singlecolumn passage completely depleted the activity. Complete depletion wasalso observed with the H301 hamster kidney cell line (FIGS. 54E and54F).

Storage Conditions and SHBG Related Properties. At completion of thetwo-step isolation, the pools were stored in the presence of sufficientglycerol to prevent freezing at −20° C. In experiments not shown, theestrogen reversible inhibitor was progressively less stable withoutaddition of glycerol, calcium and/or steroid hormone. Dialysis againstbuffers without calcium is most definitely to be avoided. Freeze/thaw isvery harmful, even with calcium and DHT present. Assays of −20° C.glycerol stored CA-PS-pool II over a two year period indicated no decayin activity. Clearly, the storage conditions known to stabilizefunctional SHBG (Fernlund P and Lauren C-B (1981) J Steroid Biochem 14,545-552; Rosner W et al. (1974) Biochim Biophys Acta 351, 92-98) alsofavored retention of estrogen reversible inhibitor activity inCA-PS-pool II.

Labeled Steroid Hormone Binding to CA-PS-pool I. CA-PS-pool I wasdetermined to contain CBG by criteria cited above. Additionally, thispool was examined by Scatchard analysis for binding of tritium labeledsteroid hormones. The results are summarized in TABLE 9. The associationconstants (K_(a)) of the labeled hormones showed the ordercortisol>progesterone>>>sex steroid hormones. The K_(a) of cortisolbinding at 34° C. was 1.41×10⁹M⁻¹ that was equal to that of native ratCBG when analyzed at 4° C. (Rosner W (1990) Endocr Rev 11, 80-91).However, it was higher than the K_(a) of 5.2×10⁷ M⁻¹ for human CBGmeasured at 23° C. (Rosner W and Bradlow H L (1971) J Clin EndocrinolMetab 33, 193-198). The binding characteristics of steroids to CBG fromseveral species have been studied (Rosner W (1972) J Steroid Biochem 3,531-542). The similarity of the results herein further supports theconclusion that CA-PS-pool I contains predominantly CBG.

Labeled Steroid Hormone Binding to CA-PS-pool H. The estrogen reversibleinhibitor activity in CDE-serum correlated with the binding of tritiumlabeled sex steroid hormones. This suggested a relationship between theestrogen reversible inhibitor and SHBG. However, the K_(a) for ³H-DHTbinding to CDE-serum at 34° C. was 3.90×10⁷ M⁻¹. However, it isimportant to note that this was at least 20 times lower than that ofpurified human SHBG at 0.99×10⁹M⁻¹ for DHT or 2.2×10⁸ M⁻¹ for E₂ at 37°C. (Rosner W and Smith R N (1975) Biochemistry 14, 4813-4820). Todetermine if CA-PS-pool II possessed the same sex hormone bindingproperties as whole CDE-serum, and/or human SHBG, the next study wasconducted. Scatchard analysis of ³H-DHT binding to CA-PS-pool II wasdone at 34° C. The estimated K_(a) was 5.88×10⁷ M⁻¹. Replicates (N=3)gave a K_(a) range 4.5−10×10⁷M⁻¹. Computer analysis indicated a singleclass of binding sites although correlation coefficients wereapproximately 0.7. Similar analyses were done with ³H-E₂,³H-progesterone and ³H-cortisol. The results with all four labeledsteroids are summarized in TABLE 7. The K_(a) order wasDHT>E₂>>>cortisol>progesterone. The K_(a) for sex steroid hormonebinding to the CA-PS-pool II was similar to whole CDE-serum but 20 to50-fold lower than human SHBG. TABLE 7

TABLE 7 Summary of the Scatchard Analysis of phenyl-Sepharose pools Iand II with four labeled steroid hormones Steroid Hormone CA-PS-Pool ICA-PS-Pool II (³H-labeled) K_(d) (M) K_(a) (M⁻¹) K_(d) (M) K_(a) (M⁻¹)Cortisol 7.10 × 10⁻¹⁰ 1.41 × 10⁹ 1.89 × 10⁻⁶ 5.30 × 10⁵ Progesterone1.70 × 10⁻⁹ 5.90 × 10⁸ 7.89 × 10⁻⁶ 1.17 × 10⁵ 17β-estradiol 1.05 × 10⁻⁵9.51 × 10⁴ 2.83 × 10⁻⁸ 3.55 × 10⁷ Dihydrotestosterone 6.05 × 10⁻⁶ 1.64 ×10⁵ 1.43 × 10⁻⁸ 6.99 × 10⁷

Western immunoblotting with anti-human SHBG. The above shows that theestrogen reversible inhibitor shared immunological properties with humanSHBG. To investigate further, Western immunoblotting of CA-PS-pool IIwas done with anti-human SHBG. The results are presented in FIG. 52B.Western analysis with the anti-SHBG recognized the same four componentsseen with Coomassie Blue staining in FIG. 52A. These same fourcomponents have also been identified with whole CDE-serum using Westernanalysis with anti-human SHBG (data not shown). In Westernimmunoblotting studies not presented, anti-human SHBG did not identifyhorse serum albumin. This confirmed that the 67 kDa Coomassie Bluestained component present in the CA-PS-pool II was not 68 kDa horseserum albumin. These results provided additional support for theconclusion that albumin is not the estrogen reversible inhibitoractivity of serum. These results also very clearly demonstrated that theSHBG used to raise antibodies in rabbit had not been purified tohomogeneity, but rather had been used at a more “crude” state. (In apersonal communication, it was also confirmed by the manufacturer of theanti-SHBG antibody that the SHBG fraction used for antibody productionwas not highly purified and had not been size fractionated.)

Discussion of Example 15. There has been one very critical problem withthe estrocolyone hypothesis. Estrocolyone has never been purified andshown to act as described (Soto A M and Sonnenschein C (1987) Endocr Rev8, 44-52). The active pool isolated from the two-step procedure (i.e.CA-PS-pool II) certainly does not bind steroid hormones with sufficientaffinity to act as estrocolyones (TABLE 7). Growth is activated atpicomolar concentrations while the affinity (Kd) of E₂ with CA-Pool IIis about 10⁻⁸M. This discrepancy is simply far too large to accept therole of estrogens in growth as binding the inhibitor and therebypreventing its action on target cells (Soto A M and Sonnenschein C(1987) Endocr Rev 8, 44-52). The fact that proteins in CA-PS-pool IIbind steroids is not germane to the mechanism of action of thesehormones in growth regulation under physiological conditions.

The results of steroid hormone binding may however be germane to the useof high dose treatments of breast cancer. Care must be taken whenconsidering that high doses of estrogen, androgen, progesterone andcortisol all have the potential for binding the active agent inCA-PS-pool II and therefore may reduce the effective concentration ofinhibitor. The assays described in this Example can be applied tobiological fluids and plasma to determine if steroid concentrations areexcessive and to evaluate proper levels with changes in treatmentregimes.

The results presented herein indicate that the proposed new model ofcell growth is a favored mechanism. Steroid hormones appear to act aspositive agents via internal high affinity receptors (e.g. ERγ) whereasserum-borne inhibitors act at the surface to block growth. Thecombination of the two signals dictates cell proliferation rates. Thisdata further supports the assertion that the ERγ can be used fordiagnostic purposes in ER⁺ cancers, preferably in the same way thatconventional ER receptor screening is now performed.

A highly enriched fraction of serum protein was prepared whose estrogenreversible inhibitory activity is stable and whose effects replicatethose seen with full serum with a variety of sex steroid hormone targettumor cell types in culture. Because early studies mistakenly indicatedthat the inhibitor shared various properties with SHBG, a two-stepcortisol-agarose affinity and phenyl-Sepharose chromatography protocolwas applied. A highly enriched “SHBG-like” preparation was obtained. At10 to 15 μg/mL, it replicated the E₂ reversible inhibition caused by 30to 50% (v/v) serum with steroid responsive human breast cancer cells,and responsive rat mammary, rat pituitary and Syrian hamster kidneytumor cells in culture. The inhibitor retained full activity for morethan one year when stored unfrozen at −20° C. in the presence ofcalcium, dihydrotestosterone and glycerol. This study demonstrated thatthe longstanding problem of inhibitor stability has been overcome andthat a high specific activity preparation was now available to furtherprobe molecular identity. These results clearly differentiate thisinhibitor preparation from any previously described type of estrogenreversible inhibitor (i.e. estrocolyone). Moreover, no previousinhibitor composition, at a concentration≦15 μg/mL, can supplant theeffects of full serum to give estrogenic effects≧3 CPD with several ER⁺cell lines from different tissues and different species.

The most active inhibitor preparation obtained in this study appeared tohave multiple components present. The separation and identification ofthese components would yield additional assays and preferred reagentsand methodologies for testing new hormone-like and anti-hormone likesubstances. The results in FIG. 52 suggest that there may be more thanone inhibitor. The active serum-derived inhibitor fraction can be useddirectly in tests of new compounds, substances, mixtures andpreparations from natural and synthetic sources to estimate bothestrogenic and androgenic activity in culture. Large-scale preparationof this purified serum fraction is possible by using larger affinitycolumns and proportionately increased serum volumes, similar to existingtechnology employed for purifying other biological products. It isadvantageous that only, small quantities of the purified serum fractionare needed for cell growth

Example 16 Serum-Free Assay Systems for Measuring Large MagnitudeSteroid Hormone Mitogenic Responses with the Two-Step Purified Inhibitor

The above-described studies with several different sex steroid sensitivecell lines demonstrated that the effects of a partially purifiedestrogen reversible inhibitor could readily be assayed in the presenceof a low concentration (i.e. 2.5%) of CDE-serum. The next step was toeliminate the serum completely and to show estrogen responsiveness underfar more defined conditions.

Second Analysis of Serum-free Growth±E2. Experiments were conductedusing completely serum-free medium, and the magnitude of the estrogeniceffects observed in defined medium was again compared to those seen inmedium containing CDE-serum. ER⁺ tumor cell growth was measured first inserum-free defined culture±10 nM E₂. Similar experiments have beenreported in FIGS. 47 and 48. The new assays were included here becausethe first experiments were done two years earlier. The results show thestability of the cell lines used and the fact that serum-free definedmedium is highly reproducible. More recent results are shown with theMCF-7K human breast cancer cells (FIG. 55A), the T47D human breastcancer cells (FIG. 55B), the GH₄C₁ rat pituitary tumor cells (FIG. 55C),and the H301 Syrian hamster kidney tumor cells (FIG. 55D). All four-celllines grew logarithmically for several days in defined and reacheddensities of 0.5 to 1.0×10⁶ cells per 35-mm dish. The media formulationswere based on standard D-MEM/F-12 as described in TABLE 6. Growth rateswere optimized to 70% or more of D-MEM/F-12 containing 10% (v/v) fetalbovine serum. The results presented in FIG. 55 show little or no E₂effect on growth in defined medium. Barnes and Sato (Barnes D and Sato G(1980) Nature (Lond) 281, 388-389) have reported similar negativeresults with another strain of MCF-7 cells in a different formulation ofdefined medium. Considering the variety of cell types assayed herein,the present results and the results of others, the lack of estrogeniceffects in serum-free defined medium was not related to chemicalcomposition of any one medium nor was there a major problem with timedependent variation of cell line properties.

Effects of CDE-Serum on ER⁺ Cells in Different Formulations ofSerum-free Defined Medium. The experiments in FIG. 56 were done to showthat serum could be added different formulations of defined medium andstill cause estrogen reversible inhibition. Effects are shown withCDE-horse serum±10 nM E₂ and T47D cells DDM-2MF (FIG. 56A), MTW9/PL2cells in DDM-2A (FIG. 56B) and GH₄C₁ cells in PCM-9 (FIG. 56C).Definitely, the serum-borne inhibitor(s) was fully effective in threedifferent formulations of defined medium and with three differentestrogen target tissue cell types.

Effects of CA-PS-pool II on ER⁺ Cell Growth in Serum-free DefinedMedium. The estrogen reversible inhibitory effects of CA-PS-pool II wereexamined with eight ER⁺ cell lines growing in different serum-freedefined media (FIG. 57). The cell lines were the MCF-7K cells (FIG.57A), the T47D cells (FIG. 57B), the ZR-75-1 human breast cancer cells(ATCC) (FIG. 57C), the GH₁ (ATCC) (FIG. 57D), GH₃ (ATCC) (FIG. 57E), andGH₄C₁ (FIG. 57F) rat pituitary tumor cells, the MTW9/PL2 rat mammarytumor cells (FIG. 57G), and the H301 Syrian hamster kidney tumor cells(FIG. 57H). At 20 to 30 μg/mL, this fraction completely inhibitedgrowth. The inhibition was totally reversed by 10 nM E₂. The E₂ effectson cell number were in the range from 33 to 72-fold (i.e. CPD=2^(5.04)to 2^(6.18)). The activity was not replaced by serum albumin at 5 mg/mL(data not shown). The estrogen mitogenic effects seen in defined mediumcontaining only a few μg/mL of protein were equal to or greater thanthose seen in medium containing 30 to 50% (v/v) CDE-horse serum withevery ER⁺ cell line tested (TABLE 8). Plainly, the serum-free conditionsestablished herein are the most defined model assay systems yetestablished to demonstrate estrogen responsiveness in vitro.

TABLE 8 Summary of the Maximum Estrogenic Effects in D-MEM/F-12 plusCDE-horse Serum 10 nM E₂ versus those in Serum-free Defined MediumSupplemented with CA-PS-pool II MAXIMUM ESTROGENIC EFFECTS IN SERUM-FREE MAXIMUM ESTROGENIC MEDIUM PLUS CELL LINES EFFECTS IN CDE-SERUMCA-PS-POOL II MCF-7K 3.40 CPD (2^(3.40) = 10.5-fold) 5.84 CPD (2^(5.84)= 57.3-fold) T47D 5.38 CPD (2^(5.38) = 41.6-fold) 5.88 CPD (2^(5.88) =58.9-fold) ZR-75-1 3.84 CPD (2^(3.84) = 14.3-fold) 5.21 CPD (2^(5.21) =37.0-fold) GH₁ 4.71 CPD (2^(4.71) = 26.2-fold) 5.04 CPD (2^(5.04) =32.9-fold) GH₃ 4.78 CPD (2^(4.78) = 27.4-fold) 5.04 CPD (2^(5.04) =32.9-fold) GH₄C₁ 4.82 CPD (2^(4.82) = 28.2-fold) 5.11 CPD (2^(5.11) =34.5-fold) MTW9/PL2 6.22 CPD (2^(6.22) = 74.5-fold) 6.18 CPD (2^(6.18) =72.5-fold) H301 4.33 CPD (2^(4.33) = 20.1-fold) 6.01 CPD (2^(6.01) =64.4-fold) CPD (2^(CPD) = Fold Cell Number Increases Above ControlsWithout Estrogen)

Discussion of Example 16. The studies presented in FIG. 57 and TABLE 8summarized unequivocally, and for the very first time, demonstrate thatlarge magnitude estrogen mitogenic responses can be observed incompletely serum-free defined media containing 2 mg/mL total protein.Furthermore, the responses shown in FIG. 57 either equal or exceedothers previously observed in partially serum-free media with ZR-75-1human breast cancer cells (Allegra J C and Lippman M E (1978) Cancer Res38, 3823-3829; Darbre P D et al. (1984) Cancer Res 44, 2790-2793) orwith a variety of other estrogen sensitive (ER') human and rodent celllines in medium with hormone depleted or deficient serum (Amara J F andDannies P S (1983) Endocrinology 112, 1141-1143; Natoli C et al. (1983)Breast Cancer Res Treat 3, 23-32; Soto A M et al. (1986) Cancer Res 46,2271-2275; Wiese T E et al. (1992) In Vitro Cell Dev Biol 28A, 595-602).

These results have a number of important implications, one of which isthat they support the aspect of the estrocolyone hypothesis (Soto A Mand Sonnenschein C (1987) Endocr Rev 8, 44-52) that relates to thepresence in serum of a meaningful inhibitor(s). Also, in view of thepresent results, there is no doubt that the inhibitor(s) is/arecompletely estrogen reversible. However, the present experimentalresults do not confirm that the steroid hormones interact with theinhibitor with sufficient affinity to support that aspect of theestrocolyone hypothesis. The results in TABLE 7 indicate that thissteroid hormone binding aspect of the estrocolyone hypothesis is highlyunlikely.

The estrogen reversibility of the inhibitor with every target cell typestudied under the rigorous conditions of serum-free defined culturesuggests physiologic relevance. The large magnitude of the effects is astrong statement in favor of significance. This is especially clear whenconsidering the fact that the first experiments with 30 to 50% (v/v)serum contained 15 to 25 mg/mL of protein, whereas the later tests usingserum-free medium required only 20 μg/mL of isolated protein.

The active fraction isolated from horse serum represented only 0.01 to0.04% (w/w) of the total protein. Nonetheless, it effectively regulatedeight ER⁺ cell lines derived from three species and three differenttarget tissues. These observations are evidence that a broadlyapplicable serum fraction has been identified. Furthermore, theserum-free medium results suggest that a common agent(s) maycoordinately regulate estrogen responsive tissue growth in vivo and thatthe concept of estrogen reversible negative control may be far-reaching.The results support the conclusion that in vitro studies can be used toidentify important new aspects of in vivo endocrine physiology. Theresults of the cell growth experiments in defined medium have manypractical applications. It has been demonstrated herein that a modelcell growth assay system now exists that is valuable for assessing awide variety of cell growth effects.

Cells in serum-free medium grow in response to nutrients, growthfactors, metal delivery proteins, adhesion proteins, and various classesof hormones. All of these components are mitogenic in the sense thatthey contribute to cell replication. Nonetheless, the addition of only20 μg/mL of inhibitor to block growth completely bears directly on thequestion of the progression of normal steroid target cells to fullyhormone autonomous cancers. The inhibitor preparation used herein hasthe properties of a family of tissue regulators first named “chalones”.These proposed cell regulators are water-soluble and tissue specific(but not species specific) proliferation inhibitors that are reversibleby physiologic stimuli including hormones (Bullough W S (1975) Life Sci16, 323-330; Finkler N and Acker P (1978) Mt Sinai J Med 45, 258-264).The studies presented herein support this classic concept as it appliesto sex steroid hormone target tissues. The molecular identification ofthe serum inhibitor(s) promises not only to further support the role ofestrogens as “necessary”, but also to establish that “chalone-like”entities likely are the missing “sufficient” components that account forestrogen regulation of tissue growth. The application of serum-freedefined medium conditions along with the use of a high specific activityfraction to demonstrate estrogen responsiveness in culture is unique. Itshould be noted that “chalones” have never before been identified. Theresults presented herein indicate, and in U.S. Pat. No. ______ (Atty.Dkt. No. 1944-00201)/PCT/US2001/______ (Atty. Dkt. No. 1944-00202)entitled “Compositions and Methods for Demonstrating Secretory ImmuneSystem Regulation of Steroid Hormone Responsive Cancer Cell Growth,”hereby incorporated herein by reference, that the immune system is thelong sought after source of these tissue specific inhibitors. In theseries of studies described herein, the tissues are the mucosal tissues.

Example 17 Chemical and Immunological Properties of the PartiallyPurified CA-PS-Pool II Inhibitors and Identification as IgA and IgM

This Example describes chemical and physical confirmation that thesought-after serum-borne cancer cell growth inhibitor(s) include atleast IgA and IgM.

Antibodies Against the CA-PS-Pool II Components. Preparative SDS-PAGEwas done on the CA-PS-pool II fraction, and after localization of the 54kDa band, the 54 kDa band was eluted and prepared for rabbit antibodyproduction by HTI (Ramona, Calif.). The antibodies raised were verypotent and reacted with CA-PS-pool II (FIG. 58). They did not crossreact with CBG (CA-PS-pool I). However, despite great care, it wasevident that the anti-54 kDa was raised against a mixture of 67, 58 and54 kDa subunits (FIG. 58). The reaction was definitely strongest withthe 54 kDa component, but clearly identifiable with the 67 kDa and 58kDa bands as well. This apparent problem turned out to be an advantage,and allowed positive identification of the active agents in CA-PS-poolII. It was investigated whether the activity in CA-PS-pool II might havebeen isolated because of affinity for the agarose matrix rather than asa consequence of the steroid hormone ligand attached to agarose, notingfrom interpretation of unrelated studies, that agarose alone can bindimmunoglobulins and give SDS-PAGE bands at 67, 58 and 54 kDa. Therefore,it was thought possible that IgG was the estrogen reversible inhibitor.

Antibodies Against the 54 kDa Component of CA-PS-Pool H and Blocking ofthe Estrogen Reversible Inhibitor Activity. Based on the results in FIG.58, it was apparent that the 54 kDa antiserum might be used to determineif the biological activity resided in any of the 67, 58 or 54 kDa bands.The next study was done to resolve this important issue. The resultswere pivotal. FIG. 59 shows that the purified material in CA-PS-pool IIwas completely inhibitory at 20 to 40 μg/mL. Addition of even a 1:5000dilution of anti-54 kDa blocked the effect of the inhibitor. In controlstudies, rabbit pre-immune serum had no effect even at 1:100 a dilution(data not shown). It was evident that anti-54 kDa serum contained theantibody to the activity.

Anti-54 kDa Serum Recognizes Authentic Horse IgA, IgM and IgG. Next,authentic horse IgA was obtained from Accurate Chemicals, and horse IgMwas obtained from Accurate Chemicals and Custom MonoclonalInternational. The material from Custom Monoclonals was custom purifiedby an affinity method with a monoclonal antibody against horse IgM Fcand further purified by molecular sieve chromatography to be sure ofelimination of other immunoglobulins (a common problem). IgGs wereobtained from Zymed (San Francisco, Calif.), Sigma (St. Louis, Mo.) orThe Binding Site (San Diego, Calif.). The Western analysis shown in FIG.60 demonstrates these results. The results show clear cross-reactionwith 67 kDa IgM heavy chain, 58 kDa IgA heavy chain and 54 kDa IgG heavychain but no reaction with horse albumin.

Assay of Estrogenic Effects Controlled by Commercially Purchased HorseIgG, IgA and IgM in 2.5% CDE-horse Serum with MTW9/PL2 Cells. FIG. 61demonstrates that at concentrations up to 59 μg/mL, horse IgG did notcause inhibition of MTW9/PL2 cell growth in 2.5% CDE-horse serum. Therewas no significant estrogenic effect caused by IgG. FIG. 62 shows veryclearly that commercially prepared horse serum derived IgM (CustomMonoclonals), was very active. At concentrations of 20 to 50 μg/mL, IgMcompletely inhibited the growth of the MTW9/PL2 cells (i.e. <1.0 CPD).Addition of 10 nM E₂ reversed the inhibition nearly completely.Estrogenic effects of 4 to 5 CPD were seen (FIG. 62). FIG. 63 shows thesame general results with commercially prepared horse serum derived IgA(Accurate). The only apparent difference was that IgA was slightly moreeffective than IgM. These results clearly proved that the activecomponents in CA-PS-pool II were IgA and IgM. This was a clear sequenceof studies culminating in evidence supporting IgA and IgM. That theseimmunoglobulins would prove to be the inhibitor was completelyunexpected. Although these two active classes of immunoglobulins (IgAand IgM) are well-established secretory products of normal breast cells,there was no previous suggestion in the prior art that they play a rolein the negative regulation of estrogen-dependent cell growth. Theseimmunoglobulins are major proteins in milk whose hormone-related localproduction in breast tissue is well documented, and their function inthe body's secretory immune system is well known.

Alternate Methods of Obtaining Horse Serum IgG, IgM and IgA. IgG can bepurified using a Hytrap matrix, which is a mixture of immobilizedProtein A and Protein G, employing a technique described by others(Lindmark R et al. (1983) J. Immunol. Meth 62, 1-13; Kronvall G et al.(1969) J Immunol 103, 828-833; Akerstrom B et al. (1986) J Biol Chem261, 10240-10247). IgM can be obtained using a mannan binding proteinisolation method normally applied with human serum (Nevens J R et al.(1992) J Chrom 597, 247-256). However, yields are low. Another methodbased on anti-IgM immunoglobulins linked covalently to Sepharose is farmore effective. This same procedure with immobilized anti-IgAimmunoglobulins can be used to isolate IgA (Tharakan J In: AntibodyTechniques, Malik V S & Lillehoj E P, Eds, 1994, Academic, Press, SanDiego, Calif., Chapter 15). Horse IgA can also be purified using animmobilized Jacalin lectin method usually reserved for human samples(Roque-Barreira M C et al. (1986) Braz J Med Biol Res 19, 149-157).However, it can be modified for non-human species. The buffers aremodified to contain 10 to 50 mM CaCl₂ to bind IgA from other species.Even then, yields are not high. The preferred methods for horse IgA andIgM use immobilized antibodies.

Purification of Rat Serum Immunoglobulins. Three isolations of theestrogen reversible inhibitor from separate one-liter batches of adultrat serum were conducted. This was done for two important reasons.First, the estrogen reversible activity in all types of adult serum,including rat, were assayed with a highly estrogen sensitive MTW9/PL2rat mammary tumor cell line. It was useful to confirm the horse serumpurification results with a homologous experimental system. Second, theconfirmation that rat IgA and IgM regulated rat mammary tumor cellgrowth would open the possibility of combined testing of new therapeuticsubstances both in vitro and in vivo. To summarize, the same “CBG” and“SHBG” fractions were obtained from rat serum by the methods of Fernlund& Laurell as had been obtained from horse serum. The chromatographyprofiles of the rat separations (not presented) were very similar tothose presented in FIG. 51. The only major difference was that with ratserum, the first peak (i.e. CA-PS-pool I) contained no CBG. At pH 5.5,rat CBG did not significantly bind to the affinity matrix. Rat serumCA-PS-pool I and CA-PS-pool II both contained only two Coomassie Bluestained bands when analyzed by SDS-PAGE (FIG. 64A). These wereapproximately 55 kDa and 54 kDa. They were somewhat lower molecularweights than found with horse, and there were fewer bands. To test ifeither rat band was IgG, a Western analysis was performed with rabbitanti-rat IgG (FIG. 64B). The antibody did not recognize the Coomassiestained bands but did react with control IgG. However, when examinedwith very specific heavy chain monoclonal antibodies raised to rat IgG1,IgA, and IgM (purchased from Zymed), the Western analysis was clear(FIG. 65). Both the commercially purified rat immunoglobulins (purchasedfrom Zymed) and the two-step purified pools showed cross-reaction withanti-IgA (weakly), anti-IgG1 subtype (strong reaction) and anti-IgM(moderate reaction) (FIGS. 65A, 65B, 65C, respectively).

Rat and Horse Serum Active Pools Isolated by the Two-Step Procedure ofFernlund and Laurell have the same Classes of Immunoglobulins. The sameclasses of immunoglobulins obtained by the two-step procedure ofFernlund and Laurell (Fernlund P and Laurell C-B (1981) J SteroidBiochem 14, 545-552) with horse serum were found when rat serum was thestarting material. This was considered to be further confirmation thatbinding to the agarose matrix was more important than to the immobilizedcortisol. It should be noted that in the original Fernlund and Laurellreport using human cord serum does not address possible immunoglobulincontamination, however (Fernlund P and Laurell C-B (1981) J SteroidBiochem 14; 545-552). This is particularly curious because humanimmunoglobulins bind to agarose (Smith R L and Griffin C A (1985)Thombosis Res 37, 91-101).

Labeled Steroid Hormone Binding to The “SHBG-like” Pools from Rat Serum.As described in TABLE 6, CA-PS-pool II from horse serum binds sexsteroids with an affinity of about 10⁻⁸ M. This same Scatchard analysiswas done with an active fraction from rat serum. TABLE 9 shows theresults of these studies with four labeled steroid hormones. It is clearthat sex steroid hormones bind with a higher affinity than progesteroneor cortisol. The binding affinities of rat and horse preparations werevery similar. In both cases, the affinities tend to rule out theestrocolyone hypothesis because it requires E₂ binding in the picomolarrange.

TABLE 9 Summary of the Scatchard Analysis of the “SHBG-like” Pools fromRat Serum with Labeled Steroid Hormones Steroid Hormone CA-PS-Pool II(3H-labeled) K_(d) (M) K_(a) (M⁻¹) Cortisol 5.7 × 10⁻⁶ 1.8 × 10⁵Progesterone 6.9 × 10⁻⁶ 1.4 × 10⁵ 17β-estradiol 4.1 × 10⁻⁸ 2.4 × 10⁷Dihydrotestosterone 2.4 × 10⁻⁸ 4.1 × 10⁷

Evaluation of the Rabbit Anti-SHBG Cross-Reaction with the Active Poolsfrom the Two-Step Isolation of Fernlund and Laurel. As shown above inFIG. 52B, Western analysis with the anti-SHBG detected horse IgA, IgMand IgG. Additionally, anti-SHBG immunoprecipitated the estrogenicactivity of horse serum (results not presented): To extend theseresults, it was established that rabbit anti-human SHBG recognized anumber of the major classes and subclasses of rat immunoglobulins.SDS-PAGE with Coomassie blue staining (FIG. 66A) was compared toidentification of the same proteins by Western analysis with anti-SHBG(FIG. 66B). These results leave very little doubt that the human plasmaderived SHBG used to raise antibodies in rabbits was not homogeneous butin fact was a “crude” preparation contaminated with severalimmunoglobulins.

Test of Rat IgG, IgA and IgM for Estrogen Reversible Inhibitory Activitywith MTW9/PL2 Rat Mammary Tumor Cells. All of the rat immunoglobulinsdescribed in this section were purchased from Zymed as the highestquality available. Their activity was assessed with MTW9/PL2 cells in2.5% (v/v) CDE-rat serum, as described above. The activity of rat IgG(all subclasses combined) was assessed (FIG. 67). There was noinhibitory effect at up to 50 μg/mL. Rat IgA was a potent estrogenreversible inhibitor (FIGS. 68). At 20 to 50 μg/mL, it completelyinhibited growth. Addition of 10 nM E₂ completely reversed theinhibition. The estrogenic effects recorded were >5 CPD. The resultswith rat IgM were very similar (FIGS. 69). At 20 to 50 μg/mL, itcompletely inhibited growth. Addition of 10 nM E₂ reversed theinhibition. The estrogenic effects recorded were >5 CPD. It is essentialto note that IgA or IgM replaced the effect of full CDE-rat serum withMTW9/PL2 cells. With a completely homologous system (i.e. cell line,basal 2.5% CDE-serum, and immunoglobulins), the results were clear. IgAand IgM were the sought after serum-borne inhibitors from rat.

Discussion of Example 17. The identification of IgA and IgM asserum-borne inhibitors fully separates these inhibitors from theteachings of U.S. Pat. Nos. 4,859,585 (Sonnenschein) and 5,135,849(Soto), which arrived at no molecular identification of the inhibitor.The series of investigations described above demonstrate that a verylongstanding problem has been solved. While the solution is significant,an even more an important consequence of this knowledge is the fact thatfor the very first time, mucosal cell hormone dependent growth has beenlinked to a natural immune regulation. Moreover, this information hasdirect application to the diagnosis, genetic screening, prevention andtherapy of breast and prostate cancer and a high likelihood ofapplications to other mucosal cancers, as also described elsewhereherein.

During the purification of both the horse serum and the rat serumestrogen reversible activity, SUPERDEX™ (Pharmacia) molecular sievechromatography of the final mixtures indicated the presence of <20% 160kDa monomeric immunoglobulins. The majority of the material was of muchlarger mass. Because IgA exists naturally as monomer, dimer andpolymers, there was a question concerning which of these is/areinhibitory form(s). The SUPERDEX™ results strongly favor thedimer/polymer form. This was confirmed also with commercially preparedIgA that was obtained from hybridoma and myeloma cell lines. The IgAfrom these was >80% dimer/polymer. It was very active as an inhibitor.In light of these results, it is suggested that these forms are the“good” type of IgA in the body, and that direct measurement of theirconcentration in plasma and body fluids has diagnostic and prognosticapplications.

Test methods similar to those described above, but performed with adefined, preferably minimum serum, plus purified immunoglobulininhibitor (“inhibitor spiked serum”) provide a new approach toevaluating potentially cell growth affecting substances, mixtures andcompounds that might be influenced by serum components. For example, aserum composition might contain steroid hormone free serum, such as astandard, commercially available fetal bovine serum preparation, and apredetermined amount of an immunoglobulin inhibitor, i.e., one or moreof IgA, IgM or IgG. Testing under these conditions, with a known amountof inhibitor in the serum, may be desirable or required when thesubstance has potential for inactivation/activation by a serum componentor when it has lipophilic properties that require a minimum proteinconcentration in the medium to prevent loss.

Another valuable application of the immunoglobulin inhibitors will be inidentifying substances that may have direct effects on the action of theimmunoglobulins to cause inactivation. An assay of this nature is uniquein the sense that incubation of substances with the immunoglobulin canbe done before the assay to determine effects on natural immuneresponses. Changes in environmental/chemical factors that affect thebody's immune system are of major medical concern. They also are ofgreat concern to veterinary medicine. Chemicals/nutritional supplementsmay affect immune function of domestic animals and thereby affect humanfood supplies.

This series of investigations demonstrate at least two immunoglobulininhibitors in serum. More than one inhibitor was suggested by theconventional purification data in a preceding Example, and was provedtrue in succeeding examples. There may still be other useful estrogenreversible immunoglobulin inhibitors in serum that are yet to beidentified from serum or tissue sources. The methods described in thisExample have direct application to the search for new compounds thatmimic the effect of the immunoglobulins as estrogen reversibleinhibitors. Such application opens a new avenue of search for anticancerdrugs.

Example 18 Regulation of Steroid Hormone-Responsive and ThyroidHormone-Responsive Cancer Cell Growth in Serum-Free Defined Medium bySecretory and Plasma Forms of IgA and Plasma and Cell Culture DerivedIgM

The determination of whether purified IgA and IgM from several speciesmimicked the sex steroid hormone reversible inhibitors isolated fromhorse in serum was sought. These studies included ER⁺ tumor cellsderived from rodents as well ER⁺ and AR⁺ cells from human cancers.Completely serum-free defined culture conditions were used to performcell growth assays using the purified inhibitors. The total proteinconcentration in the media was <2 mg/mL. The estrogenic and androgeniceffects observed in these assays are unique, as like effects have notbeen achieved previously in completely serum-free defined medium.

Sources of Purified IgA and IgM. Human IgM was purified from humanplasma as described using immobilized mannan-binding protein (Nevens J Ret al. (1992) J Chromatography 597, 247-256). As an example of theeffectiveness of this isolation, FIG. 70 shows SDS-PAGE and CoomassieBlue Staining with two preparations of human plasma IgM prepared. HumanIgA1 and IgA2 were purified using immobilized Jacalin (Roque-Barreira MC and Campos-Neto A (1985) J Immunol 134, 1740-1743; Kondoh H et al.(1986) J Immunol Methods 88, 171-173; Pack T D (1999) AmericanBiotechnology Laboratory 17, 16-19; Loomes L M et al. (1991) J ImmunolMethods 141, 209-218). Rat IgA and IgM were purchased from Zymed. Theeffectiveness of the Jacalin method with human plasma is shown in FIG.71. Horse IgA and IgM were purchased from Accurate, Sigma and CustomMonoclonals. IgA and IgM from other species or as products from cellculture are purchased from Sigma or Accurate. Human IgA and IgM werebought also from Sigma and Accurate. Human secretory (milk) IgA (sIgA)was purchased from Sigma or Accurate.

MTW9/PL2 rat mammary tumor cells. For this series of experiments theserum-free defined medium was the preferred formulation of DDM-2Adescribed in TABLE 6. The cell growth assays with this cell line inDDM-2A testing increasing concentrations of human plasma IgM is shown inFIG. 72. Human plasma IgM completely inhibited growth by 20 to 60 μg/mL.The ED₅₀ was about 12 μg/mL. Based on an IgM M_(r) of 950,000, the ED₅₀concentration was 1.3×10⁻⁸ M. Complete inhibition was at 2.2×10⁻⁸ M.These concentrations are certainly within the physiological range of IgMin the plasma and body fluids such as breast milk. Based on thesestudies, a comparison was done in completely serum-free defined DDM-2Amedium of the effects of 40 μg/mL of rat plasma IgA±E₂, rat plasmaIgM±E₂, and horse plasma IgM±E₂ (FIG. 73, expressed as (A) cell numbersand (B) CPD). From the CPD calculations it was clear that no matter thespecies source, IgA and IgM were very potent estrogen reversibleinhibitors of MTW9/PL2 cell growth.

One problem occurred with the MTW9/PL2 cell assays that initially causedconcern. Human IgA was purchased from Sigma as the milk derivedimmunoglobulin. It was far less expensive than plasma IgA. For reasonsthat at first were not clear, this material was at best only partiallyinhibitory and often not inhibitory. As will be discussed below with GH₁cells, this turned out to be a significant clue to the mechanism ofaction of the immunoglobulins. Nonetheless, it is known that the heavychains of IgM and IgA from different species share primary structurehomology. This is not true of the variable regions of the light chains.The results presented support the possibility of Fc-like receptormediation of the IgA and IgM effects on MTW9/PL2 cells.

GH₁, GH₃ and GH₄C₁ rat pituitary tumor cells. For this series ofexperiments the serum-free defined medium was the preferred formulationof PCM-9 described in TABLE 6. The next serum-free defined mediumstudies were done with GH₁ cells. Example assays are shown. This cellline was highly estrogen responsive in the presence of homologous ratmyeloma derived IgA (FIG. 74). Maximum estrogenic effect was >5 CPD ormore than a 32-fold estrogen-induced increase in cell number in 10 days.A similar assay with human plasma derived IgA showed nearly the sameresults (FIG. 75). Indeed, human IgA showed greater inhibition at 10μg/mL. Another study with human IgM demonstrated that it was also anestrogen reversible inhibitor of GH₁ cell growth (FIG. 76). It was notas inhibitory as IgA with this cell line, but certainly still effective.As discussed above, in the Background of the Invention, during thesecretion process a fragment of about 80% of the poly-Ig receptor(including the five extracellular domains) becomes attached to thedimeric/polymeric form of IgA to form secretory IgA or sIgA. Thereceptor fragment is called the “secretory component”. After secretion,sIgA can be readily isolated from human milk. The effect of milk derivedsecretory IgA (sIgA) was evaluated with the GH₁ cells in PCM-9, and theresults of a representative study are shown in FIG. 77. These resultswere strikingly different than those obtained with plasma derived IgA(pIgA). SIgA was not inhibitory even at 20 μg/mL. Considering why thetwo different forms of IgA behaved so differently in the GH₁ cells, thepoly-Ig receptor was recognized as a potential candidate for themediator of the action of IgA/IgM. The poly-Ig receptor has not beenpreviously associated with any growth related function. The poly-Igreceptor is concerned with process of transcytosis of IgA/IgM, asconceptually illustrated in (FIG. 78). SIgA already has the receptorbound in the sense of the secretory piece in association with the Fcdomains of the dimer. FIG. 79 illustrates schematically the structuresof inactive monomeric IgA, the connecting or joining “J” chain, thestructure of the active dimer with “J” chain, the secretory piece orsecretory component, and the dimeric IgA structure plus secretorycomponent attached, as generally understood. The illustration shows thatthe Fc domains of dimeric IgA are blocked by the secretorypiece/component. Access to the Fc domains is required for binding to thepoly-Ig receptor.

The present series of cell growth assays above were continued with therelated GH₃ cells, again in serum-free defined the preferred formulationof PCM-9 medium. Rat myeloma derived IgA was an effective estrogenreversible inhibitor of these cells in a 9 day growth assay (FIG. 80).The maximum estrogenic effect exceeded 5. CPD. A similar assay with ratIgM was conducted (FIG. 81). It showed even greater inhibition at 10μg/mL than with IgA. The estrogenic effect recorded in 10 days wasnearly 6 CPD. These same assays were next repeated with the humanimmunoglobulins. Human pIgA was an estrogen reversible inhibitor of GH₃cell growth (FIG. 82). It was not as effective as its rat counterpart,but the estrogenic effect with the human immunoglobulin was still 4 CPD.Also, human IgM was effective with GH₃ cells (FIG. 83). Again theestrogenic effect was about 4 CPD. In the final study with GH₃ cells, itwas again apparent that human milk derived sIgA was not inhibitory (FIG.84).

The studies above with GH₁ and GH₃ cells were continued with the relatedGH₄C₁ line, again in serum-free defined PCM-9 medium. Rat myelomaderived IgA was an effective estrogen reversible inhibitor of thesecells in a 9 day growth assay (FIG. 85). The maximum estrogenic effectapproached 5 CPD. A similar assay with rat plasma IgM was conducted(FIG. 86). It showed slightly less inhibition than IgA. The estrogeniceffect recorded in 10 days was nearly 4 CPD. These same assays were nextrepeated with the human immunoglobulins. Human pIgA was an estrogenreversible inhibitor of GH₄C₁ cell growth (FIG. 87). It was not aseffective as its rat counterpart, but the estrogenic effect with thehuman immunoglobulin was still almost 4 CPD. Also, human pIgM waseffective with GH₄C₁ cells (FIG. 88). The estrogenic effect was about 5CPD. In the final study with GH₄C₁ cells it was again apparent thathuman milk derived sIgA was not inhibitory (FIG. 89).

H301 Syrian hamster kidney tumor cells. The studies with this cell linewere done in the preferred formulation of CAPM defined medium describedin TABLE 6. Because hamster IgA and IgM were not available, theseexperiments began with plasma IgA from mouse (FIG. 90). Mouse IgA wasvery effective with hamster H301 cells. The estrogenic effect was >5CPD. Human plasma IgA was also effective (FIG. 91A). The maximumestrogenic effect reached 4 CPD. Secretory IgA was inactive (FIG. 91B).With this cell line, human IgM also was an estrogen reversibleinhibitor. As shown in FIG. 92, a dose-response study demonstrated thatin serum-free defined medium with 40 μg/mL of human plasma IgM,concentrations of 0.1 to 1.0 picomolar E₂ caused significant growth(p<0.01). This data demonstrate the extraordinary sensitivity of theserum-free defined cell growth assays in the presence of immunoglobulin.The data in FIG. 92 provide strong support for the view that the H301cells can be used to characterize the new ERγ proposed in thisdisclosure. Further description of the rationale and evidence for a newgrowth regulation very high affinity estrogen receptor, ERγ, is found ina following Example.

MCF-7A and MCF-7K human breast cancer cells. For this series ofexperiments the serum-free defined medium was the preferred formulationof DDM-2MF described in TABLE 6. Two highly applied MCF-7 human breastcancer cell strains were applicable to this series of investigations. Asshown with MCF-7A cells in DDM-2MF serum-free defined medium, plasma IgAwas highly effective as an estrogen reversible inhibitor. The estrogeniceffect exceeded 4 CPD in 10 days (FIG. 93A). In contrast, sIgA wasinactive (FIG. 93B). With the MCF-7K strain, the results were nearlyidentical. Plasma IgA was effective (FIG. 94A) and sIgA was inactive(FIG. 94B). The estrogenic effects caused by pIgA were replicated bysubstitution of plasma IgM. With MCF-7A and MCF-7K, pIgM was aneffective estrogen reversible sustaining estrogenic effects of >4 CPD(FIGS. 95 and 96, respectively). In a final study of this series, an E₂dose-response experiment was conducted with MCF-7K cells in DDM-2MF plus40 μg/mL of plasma IgM. The results were remarkable. Estrogen at as lowas 0.1 picomolar caused more than one-half maximum growth response (FIG.97). The extraordinary sensitivity of this assay methodology is clearlyestablished. These results add more evidence that a very high affinityestrogen receptor (i.e. ERγ) regulates growth and is yet to be definedin human breast cancer cells.

T47D human breast cancer cells. The T47D cell line was assayed forimmunoglobulin effects in the preferred formulation of serum-freedefined medium DDM-2MF described in TABLE 6. As shown in FIG. 98A, humanplasma IgA was a very effective estrogen reversible inhibitor with T47Dcells. The maximum estrogenic effect was 6 CPD or a 72-fold cell numberincrease in 12 days. In contrast, sIgA was inactive at up to 20 μg/mL(FIG. 98B). Likewise, human plasma IgM is effective (FIG. 99),demonstrating complete inhibition of cell growth by 20 μg/mL IgM. Theestrogenic effect was 5 CPD in 12 days. In experiments not shown, theeffects of plasma derived IgM were compared to myeloma derived IgM. Thisstudy yielded the same estrogenic effects with both sources of IgM.Again, the antigenic determinant appears to be unimportant. The resultssupport the view that the heavy chains dictate the activity. In otherstudies with T47D cells in defined medium containing 40 μg/mL, thedose-response effects with E₂ showed more than one-half maximum growthat 0.1 picomolar (FIG. 100). These results continue to fortify the themethat the methods described in this Example allow investigation ofpotential estrogenic compounds and substances that might be present insamples of industrial or biological materials at very lowconcentrations. It is also apparent that the data supports the view thata high affinity ERγ regulates growth.

ZR-75-1 human breast cancer cells. For these experiments the serum-freemedium was the preferred formulation of DDM-2MF described in TABLE 6.Plasma IgA was an estrogen reversible inhibitor with ZR-75-1 cells (FIG.101A). The estrogenic effect was recorded at 5 CPD in 14 days. As seenbefore with the other ER⁺ cell lines above, sIgA was not an inhibitorwith ZR-75-1 cells (FIG. 101B). Plasma IgM was also assayed with theZR-75-1 cells (FIG. 102). It was a potent estrogen reversible inhibitorunder these completely serum-free defined conditions. As discussedabove, this line had been thought to be estrogen responsive inserum-free culture. However, the former methods were not serum-free. Asdisclosed herein, it has now been established in entirely differentculture conditions and shown that this line is truly estrogen growthresponsive in culture.

HT-29 human colon cancer cells. For this series of experiments theserum-free defined medium was the preferred formulation of CAPMdescribed in TABLE 6. As expected from endocrine physiology, colon isnot a sex steroid hormone growth regulated tissue as are others such asbreast, uterus, ovary and pituitary. However, it was discovered thatthis tissue is thyroid hormone growth responsive. As shown in FIG. 103,HT-29 human colonic carcinoma cells grow in CAPM independently of thepresence of thyroid hormone. This growth is promoted by the otherfactors present in CAPM minus T₃. However addition of plasma IgM at 40μg/mL had a dramatic effect. In the absence of T₃ HT-29 cell growth wasinhibited to ≦1.0 CPD in 10 days. Addition of increasing concentrationsof T₃ restored growth (FIG. 103). This demonstrates that colonic cancercells respond to thyroid hormones in the same manner that ER⁺ cellsrespond to E₂. Estrogens and thyroid hormones belong to the samesuperfamily of receptors and both are required for normal physiologicgrowth and development (Williams G R and Franklyn J A (1994) BaillieresClin Endocinol Metab 8, 241-266; Tsai M J and O'Malley B W (1994) AnnuRev Biochem 63, 451-486). This is the first demonstration of a secretoryimmunoglobulin acting directly as a thyroid hormone reversible growthinhibitor of a human origin colon cancer cell line.

LNCaP human prostatic carcinoma cells. For this series of experimentsthe serum-free defined medium was the preferred formulation of CAPMdescribed in TABLE 6. LNCaP cells were negatively regulated by plasmaIgA (FIG. 104A). The immunoglobulin was a DHT reversible inhibitor thatwas completely effective at 10 μg/mL. The androgenic effect was >5 CPDin 12 days. As seen with the ER⁺ cell lines above, sIgA was notinhibitory with LNCaP cells (FIG. 104B). Two different types of humanIgM were also compared with LNCaP cells (FIG. 105). They were plasmaderived and myeloma derived IgM. Despite the differences in antigenbinding domains, both forms were equally inhibitory and both forms werereversed by 10 nM DHT. These results indicate that the Fe/heavy chain ofIgM is the functional activator of the inhibition.

Summary of the estrogenic effects of IgM on ER⁺ cell growth. FIG. 137presents a summary of the effects of IgM derived from different specieswith a variety of ER⁺ cell lines. This summary presents the maximumestrogenic effects recorded under conditions described above inserum-free defined medium with each cell line±10 nM E₂. Estrogeniceffects ranged from 4 to >7 CPD. Comparison of the results in FIG. 106with those in TABLE 8 show in general that the results achieved incompletely defined medium are equal to or greater than those seen inCDE-serum cultures.

Discussion of Example 18. These methods will permit evaluation ofindustrial, environmental, biological, medical, veterinary medicine andother potential sources of estrogenic or androgenic activity under themost sensitive conditions yet developed. Estrogenic activity ismeasurable at <1.0 picomolar concentrations. Two cell lines, MTW9/PL2and H301, are preferred potential sources of identification of the newgrowth regulatory ERγ. The evidence presented with MCF-7 and T47D humanbreast cancer cells support the presence of a new growth regulatory ERγ.The serum-free methods described herein provide unique tools to searchfor ERγ. Assays conducted under these conditions permit estimation ofestrogen sensitivities in ranges not approachable by other technology.These methods can also be adapted to measurement of the inhibitor inbiological fluids available in only small supply. For example, coupledwith use of XAD-4™ resin extraction to remove steroids, bodily fluidsand other source materials can be assayed on small scale to determinethe concentration of effective inhibitor. This is of particular interestbecause IgA in plasma is >90% inactive monomer and <10% activedimer/polymer. Measurement of IgA by conventional methods gives totalconcentrations, and does not determine the concentration/presence ofactive inhibitor. The present biological activity method has distinctfeatures and advantages, and can serve as an adjunct measurement.

The serum-free defined medium assays described herein can be used tosearch for new compounds that mimic the action of immunoglobulins toblock cancer cell growth in its early stages. This screening can be doneunder conditions in which serum proteins might interfere. Compoundsso-identified can next be evaluated by addition of CDE-serum or XAD-4™treated serum to determine if serum proteins interfere and to determinedrug efficacy in vitro under both serum-free defined medium conditionsand serum supplemented conditions. Serum-free defined medium methods canbe used for screening of compounds that may either enhance or inhibitimmune function at the epithelial cell level. Compounds with theseactivities may have utility as immune enhancers to help reduce the riskof cancer development. These assay methods offer a screening tool forsuch compounds that has not been available before. Larger magnitudeeffects permit greater accuracy with the new assay methods whenestimating effects of substances that are less potent than naturalestrogens.

Example 19 A New High Estrogen Affinity Growth Regulating EstrogenReceptor (ERγ)

This Example provides evidence of a never before recognized receptorthat mediates estrogen responsive cell growth, and discusses potentialapplications for the receptor as a diagnostic and prognostic tool.

Steroid Hormone Superfamily of Receptors. Estrogens, androgens,progestins, corticosteroids, mineral steroids, vitamin D, retinoic acidand thyroid hormone receptors all belong to a family of DNA bindingintracellular receptors that are activated by binding of the appropriatehormone/ligand (Evans R M (1988) Science (Wash DC) 240, 889-895; GiguereV (1990) Genetic Eng (NY) 12, 183-200; Williams G R and Franklyn J A(1994) Baillieres Clin Endocrinol Metab 8, 241-266; Kumar R and ThompsonE B (1999) Steroids 64, 310-319; Pemrick S M et al. (1994) Leukemia 8,1797-806; Carson-Jurica M A et al. (1990), Endocr Rev 11, 201-220; TsaiM J and O'Malley B W (1994) Annu Rev Biochem 63, 451-486; Alberts B etal. (1994) Molecular Biology of The Cell, 3rd edition, GarlandPublishing, New York, pp 729-731). The estrogen receptor described inthe citations above is now designated the classical estrogen receptoralpha (ERα). Its role in steroid regulated gene expression has beenstudied extensively and often reviewed (Yamamoto K R (1985) Annu RevGenet. 19, 209-252; Green S and Chambon P (1991) In: Nuclear HormoneReceptors, Academic Press, New York, pp 15-38; Tsai M-J and O'Malley B W(1994) Annu Rev Biochem 63, 451-486; McDonnell D P et al. (1992) ProcNatl Acad Sci USA 89, 10563-10567; Landel C C et al. (1994) MolEndocrinol 8, 1407-1419; Landers J P and Spelsberg T C (1992) Crit. RevEukary Gene Exp 2, 19-63; Cavailles V et al. (1994) Proc Natl Acad (SciUSA 91, 10009-10013; Halachmi S et al. (1994) Science (Wash DC) 264,1455-1458; Brasch K and Ochs R L (1995) Int rev Cyto 159, 161-194; HärdT and Gustafsson J-Å (1993) Acc Chem Res 26, 644-650).

Human Mutation and Mouse Knock-out Studies of ERα and ERβ. It isnoteworthy that estrogen resistance in man is caused by a mutation inthe ERα (Smith E P et al. N Eng J Med 331, 1056-1061). The moststartling fact is that this point mutation (i.e. cytosine→thymidine)generated a premature stop codon, but was not lethal. Although manymetabolic abnormalities were noted, development into adulthood wasobserved without expression of a functional ERα. This fact is furtherstrengthened by the experiments with ERα gene knockout mice (Couse J Fand Korach K S (1999) Endocr Rev 20, 358-417). Those authors state “thelist of unpredictable phenotypes in the α ERKO (estrogen receptorknockout) must begin with the observation that generation of an animallacking a functional ER α gene was successful and produced animals ofboth sexes that exhibit a life span comparable to wild-type”.Furthermore, in the review of the ERKO results it was not possible toconclude that the ERα, regulated estrogen responsive cell growth.Indeed, functions normally ascribed to the ERα seemed unaffected. Infact, only development in tissues such as breast seemed best correlated(Boccchinfuso W P and Korach K S (1997) J Mammary Gland Biol Neoplasia2, 323-334). The situation with ERKO mice and ERβ is similar (Couse J Fand Korach K S (1999) Endocr Rev 20, 358-417). The results from ERβknockout suggest an indirect role of this receptor via stromal tissue(Gustafsson J-Å and Warner M (2000) J Steroid Biochem Mol Biol 74,254-248). Certainly a direct growth role for ERβ in breast epithelialcells was not established. The results available from ERKO do not yetprovide confidence that either the ERα or the ERβ mediate estrogenresponsive cell growth.

ERα and Growth Regulation. There are other pertinent lines of evidencethat relate to the role of the ERα and growth. The first is from a studyof transfection of estrogen receptor negative cells with the full lengthfunctional ERα (Zajchowski D A et al. (1993) Cancer Res 53, 5004-5011).The investigators arrived at a remarkable result. They had expected toregain estrogen responsive growth in the transfected hormone independentcells. This was definitely not the case. Instead, addition of E₂ causedcell growth inhibition. The results indicated that ERα was not apositive mediator, but instead a negative regulator. However, similarlytransfected estrogen responsive cell lines such as MCF-7 and T47D werenot E₂ inhibited.

As previously mentioned herein, considering the results of the presentinvestigations, it is concluded that another positive acting ER existsin the MCF-7 and T47D cells and that its function is dominant andsustains growth related gene expression even with the inhibitory ERαpresent. The existence of two ER receptors is also indicated in an olderstudy of the growth of the GH₄C₁ rat pituitary tumor cells in culture(Amara J F and Dannies P S (1983) Endocrinology 112, 1141-1143). Thoseinvestigators demonstrated a biphasic effect of E₂ on these cells. Atpicomolar concentrations, E₂ caused growth. At higher concentrations, E₂induced prolactin production secretion and inhibited growth. If tworeceptors are operating, the growth receptor is more sensitive to E₂whereas the ER regulating gene expression (e.g. prolactin mRNAproduction) is activated by higher concentrations of estrogen. This samebiphasic action of estrogen on the growth of T47D human breast cancerscells has also been noted (Chalbos D et al. (1982) J Clin EndocrinolMetab 55, 276-283). Low concentrations promoted growth, whereas higherlevels were inhibitory. Indeed, a biphasic effect also was noted withthe MCF-7 cell line (Soto A M and Sonnenschein C (1985) J SteroidBiochem 23, 87-94). When this observation is coupled with the clearstatements of Soto et al (Soto A M et al. (1986) Cancer Res 46,2271-2275) that “the free estradiol levels needed for maximum responseare significantly lower than estrophilin (i.e. ERα) K_(d)s”, there isfurther support for the view that an ER exists that regulates growth andis more estrogen sensitive (i.e. lower K_(d)) than the classical ERα.While those investigators conclude that the results exclusivelysupported their estrocolyone hypothesis, and excluded ERα as thepositive growth regulator, there was no recognition of the possibilityof a much higher affinity receptor different than ERα. Finally, there isone other issue that has perplexed endocrinologists and cancerbiologists for several years. Breast cancer is sometimes treatable withhigh doses of estrogen (Segaloff A (1981) Banbury Report 8, 229-239). Ifthe ERα is the only growth mediator, one is forced into many otherpostulates to explain this observation (Reese C C et al. (1988) Ann NYAcad Sci 538, 112-121). Indeed, it may be that full occupation of ERα isinhibitory and that another receptor is the positive signal. One otherissue that is of special interest with regard to the ERα is the factthat many tissues are known to express ERα but are not growth responsiveto estrogen. Instead, estrogens cause tissue specific gene expression.Considering the results of the present investigations, it is proposedthat those tissues lack ERγ, and are therefore not growth responsive.

Variant Estrogen Receptors. Certain variant estrogen receptors have beenidentified recently by others. For example, from the estrogen growthresponsive T47D human breast cancer cell line, there have been threeisoforms of the ERα identified in one study (Wang Y and Miksicek R J(1991) Mol Endocrinol 5, 1707-1715) and another three in a differentstudy (Graham M L et al (1990) Cancer Res 50, 6208-6217). With anothertwo estrogen growth responsive human breast cancer cells lines, theMCF-7 and ZR-75-1, another ERα variant was identified that lacked theentire exon 4 of the receptor (Pfeffer U et al. (1993) Cancer Res 53,741-743). Variant receptors have also been identified from human breastcancer biopsy specimens (Murphy L C and Dotzlaw H (1989) Mol Endocrinol3, 687-693). Another truncated variant of ERα acts as a naturalinhibitor of the action of the wild-type ERα (i.e. unchanged receptor)(Fuqua S A et al. (1992) Cancer Res 52, 483-486). Another type ofvariant has received wide attention because it has constitutivetranscriptional activity without the steroid hormone ligand bound (FuquaS A et al. (1991) Cancer Res 51, 105-109). Even normal human breastepithelial cells show several natural variants of ERα (Yang J et al.(2000) Endocrine 12, 243-247). When all of these results are consideredas a group, it is clear that different forms of the ERα are possible incells, and it is reasonable to conclude that an alternate form of ERα,possibly formed by alternate splicing, or possibly arising from an asyet unrecognized gene, may regulate estrogen dependent/responsive tumorcell growth.

Characterization of ERβ. More recently, another estrogen receptor hasbeen cloned and cDNA sequenced from rat prostate and ovary (Kuiper G Get al. (1996) Proc Natl Acad Sci USA 93, 5925-5930). It has now alsobeen cloned from mouse (Tremblay G B et al. (1997) Mol Endocinol 11,353-365) and human (Mosselman S et al. (1996) FEBS Lett 392, 49-53).This new receptor has been named estrogen receptor beta (ERβ). Evidencethat ERβ is separate from ERα comes from the fact that the genes arelocated on different chromosomes (Enmark E et al. (1997) 82, 4258-4265).Therefore, ERβ is not simply an alternate splicing product of the ERαgene. Furthermore, ERβ is distinguishable from ERα based on criticaldifferences in the amino acid sequences of functional domains (Kuiper GG et al. (1996) Proc Natl Acad Sci USA 93, 5925-5930; Enmark E et al.(1997) 82, 4258-4265; Dickson R B and Stancel G M (2000) J Natl CancerInst Monogr No. 27, 135-145). For example, the sequence homology betweenthe two receptors is 97% in the DNA binding domain, but 59% in theC-terminal ligand-binding (i.e. steroid hormone-binding) domain, andonly 17% in the N-terminal domain. The ERβ N-terminal domain is muchabbreviated compared to the ERα (Enmark E et al. (1997) 82, 4258-4265).Rat ERβ contains an 18 amino acid insert in the domain binding theligand. Despite the significant differences in structure, ERα and ERβbind E₂ with the same affinity (Kuiper G G et al. (1996) Proc Natl AcadSci USA 93, 5925-5930; Dickson R B and Stancel G M (2000) J Natl CancerInst Monogr No. 27, 135-145). In fact, others (Tremblay G B et al.(1997) Mol Endocrinol 11, 353-365) have stated that ERβ has a slightlylower affinity for E₂ than ERα (Tremblay G B et al. (1997) MolEndocrinol 11, 353-365). Therefore, it is important to note that ifeither of these receptors mediates estrogen-induced growth, the steroidhormone concentrations required for one-half maximum growth (i.e. ED₅₀),or for optimum growth (i.e. ED₁₀₀), are expected to be about the same.The issue of estrogen concentrations for growth required for ED₅₀ versusthose required for one-half maximum saturation of the receptors (i.e.the dissociation constant K_(d)) will be further discussed in Examplesthat follow.

ERα and ERβ Interrelationships. Some investigators have suggested thatERα and ERβ are functionally interrelated (Kuiper G G et al. (1998)Endocrinology 139, 4252-4263) and that one role of ERβ is to modulatethe transcriptional activity of ERα (Hall J M and McDonnell D P (1999)Endocrinology 140, 5566-5578). Clearly however, there are significantfunctional differences between ERα and ERβ. These have discussed(Gustafsson J-Å (1999) J Endocrinol 163, 379-383). Also, there arefunctional differences expected because of the different pattern ofsteroid hormone binding shown by ERβ (Kuiper G G et al. (1996) Proc NatlAcad Sci USA 93, 5925-5930). For example, ERβ binds androgens whereasERα does not. This fact, plus the location of ERβ in prostate indicatesa new function that may be androgen related.

Estrogen Related Orphan Receptors. There is also another dimension ofthe estrogen receptor literature that deserves special comment. Therehave been “estrogen related receptors” (ERR 1 and 2) or “orphan”receptors identified that share properties with ERα but do not have aknown function and do not have a known ligand (Giguere V et al. (1988)Nature (Lond) 331, 91-94; Gustafsson J-Å (1999) J Endocrinol 163,379-383). Whatever mechanism is proposed for the action of the steroidhormone (i.e. on growth), it can be seen from the data presented herein,and subsequently reported elsewhere (Sirbasku D A and Moreno-Cuevas J E(2000) In Vitro Cell Dev Biol 36, 428-446), it takes a significantperiod to reverse the effects of the inhibitor. This process cannot besimply due to a rapid effect on transcription caused by steroid hormones(e.g. via a known estrogen receptor). Cellular metabolic events,including the transformation of E₂ to an active steroid metabolite, mayprovide the growth regulating ligand for one of the “orphan” estrogenreceptors. An alternative possibility is that the receptor may beactivated by metabolites formed from cholesterol metabolism (GustafssonJ-Å (1999) Science (Wash DC) 284, 1285-1286). In fact, today, there aremore than 70 “orphan” receptors seeking ligands and functions(Gustafsson J-A (1999) Science (Wash DC) 284, 1285-1286).

Comparison of the Labeled E₂ Binding Dissociation Constants (K_(d)) ofSeveral Estrogen Sensitive Cell Types. Clearly, the assays with extractsmeasured the same affinity binding sites as analyses with whole cells.This offers reasonable evidence that the standard binding technologyemployed in these studies is measuring the most common form of receptorpresent in cells, no matter whether whole cells are assayed or cellextracts. The affinity of the MTW9/PL2 estrogen receptor is that whichis characteristic of the ERα. The K_(d) of the receptor measures theconcentration of ligand that one-half saturates the sites. In TABLE 10,the K_(d) values for labeled E₂ are presented as reported and presumablyrepresent the ERα. Only when the measurements are specific for the βform is the designation (ERβ) included.

TABLE 10 Comparison of E₂ Binding Affinities Expressed as DissociationConstants (K_(d)) WHOLE CELLS CELL EXTRACTS CELL LINES K_(d) for E₂K_(d) for E₂ REFERENCES MTW9/PL2 2.78 × 10⁻⁹ M 1.89 × 10⁻⁹ MMoreno-Cuevas JE and Sirbasku DA (2000) In Vitro Cell Dev Biol 36,410-427 MCF-7 0.58 × 10⁻⁹ M 1.77 × 10⁻⁹ M MacIndoe JH et al. (1982)Steroids 39, 247-258 MCF-7-Mason 4.0 × 10⁻⁹ M Horwitz KB et al. (1978)Cancer Res 38, 2434-2437 Unfilled nuclear MCF-7-Mason × 10⁻⁹ M HorwitzKB et al. (1978) Cancer Res 38, 2434-2437 Filled nuclear MCF-7 × 10⁻⁹ MReddel RR et al. (1985) Cancer Res 45, 1525-1531 MCF-7-L 0.08 × 10⁻⁹ MMCF-7-M 0.07 × 10⁻⁹ M T47D × 10⁻⁹ M Horwitz KB et al. (1978) Cancer Res38, 2434-2437 Unfilled nuclear T47D 4.0 × 10⁻⁹ M Horwitz KB et al.(1978) Cancer Res 38, 2434-2437 Filled nuclear T47D 0.11 × 10⁻⁹ M ReddelRR et al. (1985) Cancer Res 45, 1525-1531 ZR-75-1 0.09 × 10⁻⁹ M ReddelRR et al. (1985) Cancer Res 45, 1525-1531 ZR-75-1 1.3 × 10⁻⁹ M Engel LWet al. (1978) Cancer Res 38, 3352-3364 H301  1.0 × 10⁻⁹ M Liehr JG andSirbasku DA (1985) In: Tissue Culture of Epithelial Cells, Taub M, ed,Plenum, New York, pp 205-234 H301 0.87 × 10⁻⁹ M Soto AM et al. (1988)Cancer Res 48, 3676-3680 GH₃ 0.25 × 10⁻⁹ M Moo JB et al (1982) In:Growth of Cells in Hormonally Defined Media, Vol. 9, Cold Spring Harbor,New York, pp 429-444 GH₃ 0.31 × 10⁻⁹ M Haug E et al. (1978) Mol CellEndocrinol 12, 81-95 Prostate and × 10⁻⁹ M (ERα) Tremblay GB et al.(1997) Mol Endocrinol 11, 353-365 Ovary 0.5 × 10⁻⁹ M (ERβ) Transfection0.05 to 0.1 × 10⁻⁹ M Kuiper GC et al. (1998) Endocrinology 139,4252-42-63 Studies (ERβ only)TABLE 10 presents only a fraction of the estrogen binding data availablein the literature. However, the K_(d) values presented arerepresentative and do show a discernable pattern. The lowest K_(d)identified in a literature search was in the range 5×10⁻¹⁰ M to1.0×10⁻¹⁰ M for the ERβ and 7×10⁻¹¹ M to 1.1×10⁻¹⁰ M for the ERα. Ingeneral, the binding affinities as estimated by K_(d) are lower forreceptors from human cells than those from rodent lines. It is importantto note that the results presented in TABLE 10 indicate that the lowerlimit of measuring estrogen receptor affinities most likely has beenreached. The use of the highest specific activity tritium labeledsteroids has been optimized and simply cannot be used to measure 10 to100-fold lower K_(d) concentrations. This opens the possibility of an asyet unrecognized ER that mediates growth effects at lower concentrationsof estrogen than either the ERα or the ERβ.

Discussion of Example 19. Evidence is provided herein that all of theER⁺ cell lines analyzed in this presentation show estrogenic effects(i.e. positive growth responses significant to p<0.05 or P<0.01)obtained at 10 to more than 1000-fold lower E₂ concentrations thanexpected from the measurement of K_(d), with these and related celllines. It is proposed herein that estrogen promoted growth is mediatedby an as yet to be characterized estrogen receptor designated ERγ. Inaccordance with this proposal, the ligand that activates ERγ may be E₂or another cellular component induced, changed or modified by the actionof estrogen. For example, the ligand may be a lipophilic compound suchas one of the intermediates of the cholesterol biosynthetic pathway orthe phospholipid biosynthetic pathways. There is a relationship betweenthe ERα and the ERγ, as cells that are growth responsive to estrogensexpress the ERα. This suggests that ERα has a functional, regulatory,gene, expression, or other types of control relationship to ERγ ingrowth activated target tissues. Accordingly, ERγ may be the mostaccurate estimation of breast and other cancer cell growth sensitivityto estrogens, and its measurement could serve as a valuable adjunct orreplacement for the current protocols relying on the measurement of ERαin breast cancer.

The ERγ is also suitable for application as a diagnostic and prognostictool for other cancers such as: those of the female urogenital tractincluding ovary, uterus cervix and vagina, as well as bladder, kidney,liver, melanoma, Hodgkin's disease, pituitary adenomas and other targettissues.

Example 20 Effect of Tamoxifen Antiestrogen in Serum-Free Defined Medium

In this Example the use of one of the present cell growth assays wasused to evaluate the effects of this classical antiestrogen with “mixed”functions. A new type of growth inhibiting function for tamoxifen isidentified.

Background of Tamoxifen Effects and Clinical Applications. Theantiestrogenic effects of tamoxifen are well documented. Most evidencesuggests this compound and its active metabolite 4-hydroxyl-tamoxifenprevent growth of ERα positive cells via interaction with the receptor(Coezy E et al. (1982) Cancer Res 42, 317-323; Bardon S et al. (1984)Mol Cell Endocrinol 35, 89-96; Reddel R R et al. (1985) Cancer Res 45,1525-1531). However, it has also been suggested that tamoxifen blocksgrowth factor promoted MCF-7 breast cancer cell growth (Vignon F et al.(1987) Biochem Biophys Res Commun 146, 1502-1508). Also, tamoxifen hashigh affinity binding sites and actions distinct from the estrogenreceptor (Sutherland R L et al. (1980) Nature (Lond) 288, 273-275;Phaneuf S et al. (1995) J Reprod Feral 103, 121-126). Despite itscomplex actions, tamoxifen has widespread use as a treatment for breastcancer (Fisher B et al. (1998) J Natl Cancer Inst 90, 1371-1388;Jaiyesimi I A et al (1995) J Clin Oncol 13, 513-529; Clinical TrialReport (1997) J Clin Oncol 15, 1385-1394; Clinical Trial Report (1987)Lancet 2(8552), 171-175; Forrest A P et al. (1996) Lancet 348(9029),708-713; Tormey D C et al. (1996) J Natl Cancer Inst 88, 1828-1833;Gundersen S et al. (1995) Breast Cancer Res Treat 36, 49-53; Gelber R Det al. (1996) Lancet 347(9008), 1066-1071; Raabe N K et al. (1997) ActaOncol 36, 2550260).

Serum-free Medium Effects of Tamoxifen. In the present series of tests,the effects of tamoxifen (TAM) were reexamined under completelyserum-free defined conditions. It is important to note that throughoutthe Examples herein, data is presented showing that estrogens alone haveeither had no effect on growth in defined medium or at most a 1.0 CPDeffect that was related to saturation density. This was true no matterif phenol red was present or absent from the medium, as shown herein andalso reported (Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro CellDev Biol 36, 447-464). In similar assays, 1.0×10⁻⁷ M tamoxifen wascompletely inhibitory with T47D cells in culture, as shown in FIG. 107.The study shown in FIG. 107 examined the concentrations of tamoxifenneeded to fully inhibit T47D cell growth in the preferred formulation ofDDM-2MF serum-free defined medium without any source of estrogens. Evenphenol red was eliminated: The expected outcome was no tamoxifeninhibition. As shown, estrogen alone had only a 1.0 CPD effect inserum-free defined medium. However, tamoxifen had unexpected effectsrevealed by the use of serum-free defined medium. Tamoxifen effectivelyarrested growth at 1.0×10⁻⁷ M. Higher concentrations were cytotoxic. Itwas observed in these assays that tamoxifen had the same effect asimmunoglobulins IgA and IgM. To demonstrate this fact another way, theexperimental results presented in FIG. 108 show that estrogenscompletely reversed the effect of 1.0×10⁻⁷ M tamoxifen. This sequence ofexperiments showed the same results as those shown above with plasma IgAand IgM and ER⁺ cell lines.

Discussion of Example 20, The observation of inhibition of cell growthby a classical antiestrogen demonstrates the utility of this technologyto search for other antiestrogenic compounds. Furthermore, because ofthe current intense focus on the search for SERMs (i.e. SelectiveEstrogen Receptor Modulators) the serum-free technology disclosed hereinhas particularly useful applications. Specific types of SERMS can besought for different cell types. Those SERMs that do not cause breastcancer cell growth can be readily identified by this technology. ThoseSERMs with multiple activities can be identified before conductingexpensive animal testing.

The technology presented permits a clear definition of antiestrogenswith “mixed” functions (e.g. tamoxifen-like, that act at several sites)versus those with a “pure” function mediated only by the estrogenreceptor. To date, no similar easily applied in vitro method based onserum-free defined medium and secretory immunoglobulins is availablethat produces growth as an endpoint of the assay.

An entirely new function is proposed for the well-known drug tamoxifen,in which tamoxifen mimics the immune system effects on ER⁺ cancers,thereby inhibiting growth. It is believed that estrogen reverses theseeffects, not as a consequence of interaction with the classical ERα, butas a consequence of the ERγ. This mechanism is closely parallel to thatobserved with IgA/IgM and E₂, disclosed herein. Prior to the presentinvention, tamoxifen has never been linked to growth regulatory changesin the secretory immune system, nor has there been any suggestion thattamoxifen in any way mimics the inhibitory action of IgA/IgM on mucosalcells. Accordingly, certain embodiments of the present invention offernew uses for tamoxifen based on diagnostic testing to identify humanbreast, prostate, colon and other mucosal cancers that are poly-Igreceptor/secretory component positive. For example, such identificationcould be determined by immunohistochemistry or radioimmunoassay or othersuitable tests that have clinical applicability. Those tissuesdetermined to be poly-Ig receptor/secretory component positive are thencandidates for tamoxifen treatment either alone or in conjunction withother treatment modalities. The new, preferred applications of tamoxifendescribed herein is not based on the classical ERα, which has differentcriteria for its use and different tissues as potential targets.

The serum-free assay methodology described herein will be directlyapplicable to a search for tamoxifen derivative compounds showing more“immune-like” activity or other compounds with a similar activity. Theassay method is unique because of the discovery of the estrogenreversible effects of IgA and IgM and because of the results showingthat tamoxifen inhibits in the complete absence of estrogens and isreversed by natural estrogen just as happens with IgA/IgM.

The results presented show that tamoxifen inhibits the mitogenic actionof a variety of growth factors and nutrients in completely serum-freedefined culture. This effect shows the same type of “master switch”action as demonstrated by immunoglobulins, and has mechanisticimplications. The immunoglobulins shut off all growth, as did tamoxifenin these studies. As is discussed further hereinbelow, the receptormediating the effects of the immunoglobulins must possess the propertyof a “master switch” to shut down all but steroid hormone responsivegrowth. Notably, both the immunoglobulins and tamoxifen have this effecteven when a large number of “mitogens” are present. Others have reportedthat tamoxifen inhibits growth factor dependent growth (Vignon F (1987)Biochem Biophys Res Commun 146, 1502-1508), but only concluded thattamoxifen was not a “pure” estrogen. An entirely new site of action fortamoxifen is arrived at in the present disclosure.

Tamoxifen may also be an antagonist of ERγ, and may be useful in thatcapacity. The assay methods presented herein can be used to distinguishthose antiestrogens that act only on the growth estrogen receptor fromthose acting elsewhere as well. The serum-free defined medium technologypresented herein has direct application to the assay of a great varietyof drugs now in use by women either before the onset of breast cancer orafter the onset. Drugs or preparations such as antidepressants, herbalextracts, soy products, other food, plant or microorganism extracts,estrogenic creams and cosmetic preparations can be assessed forantiestrogenic or estrogenic activity. These methodologies are alsoapplicable to exploration of additional anti-androgenic compounds.Furthermore, in view of the possible role of estrogens as well asandrogens in prostate growth, this technology can be used to search forcompounds with both activities.

Example 21 Effect of Long-Term Exposure of Breast Cancer Cells to IgMUnder Serum-Free Defined Conditions

IgM Cytotoxicity after Long-Term Exposure—MTW9/PL2 Cells. In the aboveexamples, IgM has been demonstrated to be an estrogen reversibleinhibitor of ER⁺ rodent tumor and human cancer cell growth. During thesestudies, visual inspection of the cultures indicated that experimentscarried beyond 7 days with the MTW9/PL2 cells showed a markeddeterioration of morphology. This suggested that exposure of theMTW9/PL2 cells to IgM might in fact be causing cell death. Suchobservations wee immediately recognized as having potential therapeuticapplications. To examine this further, MTW9/PL2 cells were incubated inserum-free defined medium DDM-2A for up to 10 days in the presence of 40μg/mL horse IgM (prepared by Custom Monoclonals International). On days0, 2, 4, 6, 8, and 10 after seeding, 10 nM E₂ was added to cultures andgrowth effects measured as cell number increases (FIG. 109). Theseresults, presented as cell numbers versus days, show that addition of E₂on or after day 8 no longer had an estrogenic effect. Conversion of thedata in FIG. 109 to CPD estrogenic effects showed very clearly that E₂addition after eight days no longer caused reversal of the IgM (FIG.110). The CPD after eight days with IgM were no different than thecontrols held in the absence of E₂ throughout the study. Clearly, IgMwas cytotoxic in eight days with MTW9/PL2 rat mammary tumor cells.

IgM Cytotoxicity after Long-Term Exposure—Human Cancer Cells. Similarstudies were done with the T471) and MCF-7A human breast cancer cells inserum-free defined medium DDM-2MF. Two examples are presented in FIG.111 and FIG. 112, respectively. In the presence of 40 μg/mL human pIgM,the T47D cells and the MCF-7A cells no longer responded to 10 nm E₂ byday 13. Control studies indicated the killing was IgM mediated. Theconclusion was clear. IgM was cytotoxic to human breast cancer cellswithin two weeks. In a partial replica study with LNCaP cells (resultsnot shown), human pIgA exposure for 14 days caused cell death as IgM haddone with T47D cells. These results have important therapeuticimplications.

Discussion of Example 21. The results presented are the first evidencethat exposure of breast and prostate cancer cells to IgA and IgM forperiods of two weeks or longer can cause growth inhibition leading tocell death. At present, it is not known if this represents some form ofcytotoxicity or is due to a natural process such as apoptosis. Certainlyapoptosis and cancer therapy is a dynamic current research theme,however there are no apparent previous reports in the literature relatedto IgA and IgM action on mucosal cell growth and apoptosis.

A dilemma has existed for many years regarding the frequency ofmetastasis in breast, prostate and other epithelial cancers. It wouldseem that the malignant cells should populate many more new sites muchmore rapidly than actually happens in patients. To be sure, metastasesoccur at many sites, and do occur simultaneously or nearly so. However,IgA and IgM in the plasma may act to suppress the number of disseminatedcancer cells. An implication of the results of the presentinvestigations is that cancer cells in the general circulation areexposed to the effects of IgA and IgM and therefore remain inhibited orare in fact killed. Only after they are located in relativelyinaccessible sites do they not feel the full effects of IgA and IgM, andtherefore proliferate more rapidly. One example of this is the very wellknown propensity of prostate cancers to locate in bone. This is alsotrue of breast, to a significant extent. Metastatic breast and prostatecancers are very often autonomous, consistent with the presentexperimental results. Autonomous cancer cells are not inhibited by IgAand IgM, and are, therefore, free to move in plasma and proliferate atnew sites without negative immune surveillance.

Notably, the most well known human breast cancer cell line, MCF-7, wasobtained from a pleural effusion of a patient with an estrogenresponsive cancer (Soule H D et al (1973) J Natl Cancer Inst 51,1409-1416). Indeed, many researchers have sought breast cancer celllines from this fluid. The question of why this estrogen responsive andhighly immunoglobulin sensitive line survived at this new site becomesclearer, in light of the present disclosure, when it is recognized thatplural fluid is not rich in plasma immunoglobulins. Pleural fluid is afiltrate of plasma. Elevation of plasma IgA and IgM levels may havepreventative value with regard to metastasis, and therapeutic value withrespect to those tumors that are accessible to the plasmaimmunoglobulins.

Example 22 The Role of the Poly-Ig Receptor in Hormone Responsive andAutonomous Breast and Prostate Cell Growth Regulation

In this Example it was shown that the poly-Ig receptor or a poly-Ig likereceptor mediates the inhibition of cell growth by IgA/IgM. Methods ofidentifying genetic or expression defects in that receptor, andscreening methods for assessing susceptibility, and for establishing adiagnosis or prognosis in mucosal cancers are described.

Structural Properties of the Poly-Ig Receptor. The negative response toIgA and IgM is mediated by the mucosal poly-Ig receptor or a verysimilar structure with the same immunoglobulins specificity as well asthe same immunological and M_(r) properties. The poly-Ig receptor is aM_(r) 100,000 transmembrane protein with several properties that placeit in the Ig superfamily of receptors (Kraj{hacek over (c)}i P et al.(1992) Eur J Immunol 22, 2309-2315; Williams A F and Barclay A N (1988)Annu Rev Immunol 6, 381-405). The poly-Ig receptor and the secretorycomponent from human has been cDNA cloned and DNA sequenced (Kraj{hacekover (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315; Kraj{hacek over(c)}i P et al. (1995) Adv Exp Med Biol 371A, 617-623; Kraj{hacek over(c)}i P et al. (1991) Hum Genet. 87, 642-648; Kraj{hacek over (c)}i P etal. (1989) Biochem Biophys Res Commun 237, 9-20) as has the poly-Igreceptor from mouse (Kushiro A and Sato T (1997) Gene 204, 277-282;Piskurich J F et al. (1995) and bovine tissue (Verbeet M P et al. (1995)Gene 164, 329-333). Altogether, the human poly-Ig receptor codingsequence encompassed 11 exons. The extracellular five domains originatefrom exons 3 (D1), exon 4 (D2) exon 5 (D3 and D4), exon 6 (D5), exon 7(the conserved cleavage site to form the secretory component), exon 8(the membrane spanning domain), exon 9 (a serine residue required fortranscytosis), exon 9 (sequence to avoid degradation), exon 10, no knownfunction) and exon 11 (sequence contains a threonine residue and theCOOH terminus) (Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22,2309-2315).

With the exception of domains 3 and 4 (both from one exon), the receptorstructure follows the rule of one domain/one exon. The poly-Ig receptorbinds IgA and IgM via their Fc domains, and more particularly, via aspecific amino acid sequence (15→37) of domain 1 (Bakos M-A et al.(1991) J Immunol 147, 3419-3426). Of the other extracellular domains,only D5 is known for a specific function. It contains the disulfidebonds that covalently exchange with dimeric/polymeric IgA to form sIgAduring transcytosis. The role of this receptor in transcytosis ofIgA/IgM has been well studied with mucosal tissues and epithelial cellsin culture (Vaerman J P et al. (1998) Eur J Immunol 28, 171-182; Fahey JV et al. (1998) Immunol Invest 27, 167-180; Brandtzaeg P (1997) J ReprodImmunol 36, 23-50; Loman S et al. (1997) Am J Physiol 272, L951-L958;Mostov K E et al. (1995) Cold Spring Harbor Symp Quant Biol 60; 775-781;Schaerer E et al. (1990) J Cell Biol 110, 987-998). One serine residueis particularly important for transcytosis (Hirt R P et al. (1993) Cell74, 245-255).

Lines of Evidence Supporting Poly-Ig Receptor or a Poly-Ig like Receptorin Negative Growth Regulation. A series of studies and observationsdisclosed herein indicate that the IgA/IgM inhibition mediating receptorhas the properties of the poly-Ig receptor or another receptor (“poly-Iglike receptor”) with properties very similar to those of the poly-Igreceptor. From those studies, the following supporting facts weregained: (1) The source of the active IgA is not the deciding factor.Plasma or myeloma derived IgA are equally effective. Also, species makeslittle or no apparent difference in activity. IgA isolated from variousspecies has major sequence homology in the α heavy chains butdifferences in the variable chains. This is consistent with mediation byan Fc superfamily receptor. The poly-Ig receptor is a member of this Fcbinding family. (2) IgA obtained from commercial myeloma cell sources(especially from Zymed) contains predominantly dimeric and polymericimmunoglobulin. It is highly active. This is consistent with mediationby the poly-Ig receptor because it binds only dimeric/polymeric IgA. (3)Cultures containing the active CA-PS-pool II material are 90%dimeric/polymeric forms of immunoglobulins. Experiments described hereindemonstrated clearly that this material is as active as any commerciallyprepared IgA in both serum-supplemented and serum-free defined medium.This is consistent with the expected binding of IgA to the poly-Igreceptor. (4) IgM is at least as active, or two to three times as activeas dimeric IgA, on a molar basis. Dimeric IgA is a 350 kDa complex. IgMis a 950 kDa pentamer. These masses favor IgM by two to three-fold on amolar basis. Also, IgM has five Fc domains for binding, and dimeric IgAtwo Fc domains. The source of the IgM can be from plasma or myelomacells. They are equally effective. This is expected of the poly-Igreceptor. (5) Secretory IgA is invariable inactive as an inhibitor. Ithas the five extracellular domains of poly-Ig receptor attached. Plasmaderived IgA is in contrast fully active (see FIG. 79 for IgAstructures). To prove that pIgA does not have the secretory componentwhereas sIgA contains the 801 kDa receptor fragment, the Westernanalysis in FIG. 113 was performed. Secretory IgA shows an 80 kDacross-reaction band with anti-secretory component whereas pIgA shows noreaction. This was the expected result and provides solid support forthe view that the poly-Ig receptor is the mediator. Because secretorycomponent is isolated from milk sIgA, these results show that thesecretory component used for immunization of the rabbits was free of theother subunits in IgA. This was a meaningful control for the nextexperiments.

In the next experiments, anti-human secretory component antiserum wasused to block the inhibiting effects of IgA and IgM. FIG. 114 shows theresults with the T47D cells in serum-free defined medium DDM-2MF withhuman plasma IgM alone and with a series of dilutions of the antiserum.As shown, 10 nM E₂ completely reversed the IgM inhibition. Dilutions of1:500 to 1:5000 also blocked the inhibition. In the insert in FIG. 114,it is shown that a control study with pre-immune rabbit serumdemonstrated that it had no inhibitor blocking activity. A similar studywas done with LNCaP cells in serum-free defined CAPM with human pIgA(FIG. 115). As shown, 10 nM E₂ completely reversed the pIgA inhibition.Anti-serum dilutions of 1:00 and 1:1000 also reversed the inhibition.Differences between the effective dilutions with T47D and LNCaP cellswas due to changes in lots of commercially prepared antiserum.Anti-secretory component antibodies completely blocked the inhibitoryeffects of IgA and IgM. These studies not only indicate poly-Ig receptormediation, but they support the view that IgA and IgM act via the samereceptor. The poly-Ig receptor is known to conduct transcytosis of bothof these immunoglobulins with very high efficiency.

To determine if IgA/IgM responsive cells expressed 1001 kDa poly-Igreceptor, the Western analysis shown in FIG. 116 was done. Amounts ofextracts of the designated cell types were analyzed with a 1:1000dilution of rabbit anti-human secretory component. As expected MDCKcells were positive. This cell line has been studied for several yearsas a model of poly-Ig receptor sorting and function. LNCaP cells showedthe same receptor (FIG. 116). Cell lines that were negative wereALVA-41, DU145, human fibroblasts, and PC3 cells (FIG. 116). As shown inmultiple experiments described herein, LNCaP cells are IgA/IgMinhibited. The results of the Western analyses show that they expressthe poly-Ig receptor.

In the final experiments of this series, pIgA was tested with two of thecell lines that were poly-Ig receptor negative by the Western analysisshown in FIG. 116. The results with DU145 cells are shown in FIG. 117.Plasma IgA was not an inhibitor. A similar study with PC3 cells is shownin FIG. 118. Again, pIgA was not an inhibitor even at 50 μg/mL. Theseresults demonstrate cells that lack the poly-Ig receptor are alsoinsensitive to pIgA.

The HT-29 colon cancer cells are known to express only the authenticform of the poly-Ig receptor. They are also negatively growth regulatedby IgM (FIG. 103). This implies that the poly-Ig receptor has morefunction than transcytosis only. This is very strong evidence in favorof the authentic poly-Ig receptor having a heretofore unrecognizedgrowth regulating function in early stage cancers of colon. The HT-29colon cancer cells are the source of a cDNA sequence for the poly-Igreceptor deposited in GENBANK. This sequence, hereby incorporated hereinby reference, is very often referred to in published reports and shownto be equal to the exons identified from normal human leukocytes thatwere the source of the genomic sequence of the poly-Ig receptor. Takentogether, all of the available data indicate that the authentic poly-Igreceptor has a new function, as identified and described herein.

Discussion of Example 22. For the first time, a relationship betweenimmunoglobulin growth regulation and the poly-Ig receptor isdemonstrated. This receptor has in the past been studied from theperspective of a transcytosis receptor; however, a new function for thisreceptor is now described. Gene changes in the authentic poly-Igreceptor gene may include point mutations, deletions, insertions, andpremature termination. The receptor mediating the effects of IgA/IgM maybe a form arising from alternate splicing of the original transcytosisreceptor. Changes in the regulation of expression may influence thepresence or absence of this receptor. Changes in allelic balance mayaffect the expression of this receptor and hence its function in normal,early stage cancers and in autonomous cancers. The positive correlationbetween the presence of ER and AR and expression of the poly-Ig receptorindicates regulation or positive influence by steroid hormones. Withoutwishing to be bound by a particular theory, it is suggested that thisregulation may be at the gene expression level or at another down-streamprocessing point. The actual mechanism has not yet been identified.

One of the primary themes of cancer research has been that loss of“tumor suppressor genes” causes the release of cells from negativeregulation and thereby contributes to the progression to cancer. Theevidence disclosed herein indicates that the poly-Ig receptor has a“tumor suppressor” function. It is present in cells that are regulatedby IgA/IgM and absent in cells that are insensitive to immuneinhibitors. This is a new aspect of cancer immunology that had not beenrecognized before the present invention.

For the first time, the poly-Ig receptor is connected to the D1S58linked locus that is a “hot spot” for genetic changes in breast cancer.This disclosure proposes that this locus or near neighbors contain thegrowth regulating form of authentic transcytosis poly-Ig receptor or avery similar immunoglobulin superfamily receptor. Alternately, the1q31-q41 region of chromosome 1 contains several other genes ofimmunological interest that might include the poly-Ig receptor oranother related receptor mediating the effects of IgA/IgM.

These genes are applicable for use as screens for breast and othermucosal cell cancers. They are expected to indicate susceptibility andto be used in prognosis and other diagnostic applications with humantissue and cancer samples. Analyses of allelic imbalances in thereceptor gene are also foreseen as a new tool to determinesusceptibility and prognosis for development of breast and other mucosalcancers, as will be the detection of mutations in the growth regulatingintracellular domains of the receptor. The known amino acid sequence ofthe poly-Ig receptor does not contain the immunoreceptor tyrosine-basedinhibitory motif (ITIM) common to a new family of inhibitory motifreceptors (Cambier J C (1997) Proc Natl Acad Sci USA 94, 5993-5995).Other amino acid sequences may serve this same function.

Example 23 IgG1 and IgG2 as Immunoglobulin Regulators of Estrogen andAndrogen Responsive Cancer Cell Growth

A role for IgG1 and IgG2 as immunoglobulin regulators of estrogen andandrogen responsive cancer cell growth is described in this Example,together with methods describing how to use those IgG subclasses toidentify the Fcγ receptor that mediates their inhibitory effect. Use ofthe receptor, and its gene for assessing susceptibility to cancer, andin diagnostic, gene screening and other applications is also addressed.

Background Regarding IgG Subclasses. The major immunoglobulins secretedas mucosal immune protectors include IgA, IgM and IgG. In human serum,the percent content of IgG, IgA and IgM are 80, 6 and 13%, respectively.In humans, the major subclasses of IgG are IgG1, IgG2, IgG3 and IgG4.These are 66, 23, 7 and 4% of the total IgG, respectively. The relativecontent of human immunoglobulin classes/subclasses in adult serum followthe order IgG1>IgG2>IgA1>IgM>IgG3>IgA2>IgD>IgE (Spiegelberg H L (1974)Adv Immunol 19, 259-294). When the serum concentrations ofimmunoglobulins are compared to those in exocrine secretion fluids, therelative contents change dramatically (Brandtzaeg P (1983) Ann NY AcadSci 409, 353-382; Brandtzaeg P (1985) Scand J Immunol 22, 111-146). Forexample in colostrum (a breast fluid secretion), secretory IgA is Z 80%of the total immunoglobulins. IgM is ≦10% of the total. IgG represents afew percent. In human colostrum and milk, IgG1 and IgG2 are the majorsubclasses of IgG (Kim K et al. (1992) Acta Paediatr 81, 113-118).Clearly, comparison of serum and mucosal fluid concentrations indicateselective immunoglobulin secretion. The secretion mechanism for IgA andIgM are well described. Conversely, there is a fundamental questionsurrounding IgG secretion. There is no “J” chain present in IgG1 andIgG2. From the known facts of transcytosis/secretion of immunoglobulins(Johansen F E et al. (2000) Scand J Immunol 52, 240-248), it is unlikelythat IgG secretion is mediated by the poly-Ig receptor. An epithelialreceptor specific for IgG1 has been reported in bovine mammary gland(Kemler R et al. (1975) Eur J Immunol 5, 603-608). Apparently, itpreferentially transports this class of immunoglobulins from serum intocolostrum. Despite this 1975 report however, the receptor has not beenchemically or structurally identified nor has the mechanism of transportof IgG monomers been satisfactorily defined. Certainly no growthfunction was ascribed to this “IgG1 receptor” in the 1975 Kemler et al.report. It is possible that this receptor is a member of a large groupnow designated as Fc receptors (Fridman W H (1991) FASEB J 5,2684-2690), but there is one study with IgG showing that, of 31different long-term human carcinoma cell lines, including breast, “alllines were found to be consistently Fc receptor negative” (Kerbel R S etal. (1997) Int J Cancer 20, 673-679). One possible candidate for theepithelial transport of IgG1 is the neonatal Fc receptor (Raghavan M andBjorkman P J (1996) Annu Rev Cell Dev Biol 12, 181-220). However, thereis no indication yet of the presence of this receptor in adult mucosaltissues.

Value of Assessing IgG Subclasses for Activity. Although the IgG classis lowest in concentration in secretory fluids, it is stillphysiologically important because of its capacity to neutralizepathogens by various mechanisms. The human clinical importance ofunderstanding and measuring IgG subclasses has been growing steadily.From a few clinical reports per year in 1970, the literature now exceedsfour hundred reports a year. These assays are valuable for severalreasons, including the following: (1) they provide a clearer picture ofan individual's susceptibility to disease; (2) an awareness thattreatment for subclass deficiencies is important; (3) the subclasses canbe used to assess the state of a number of diseases; and (4) the IgGsubclass difference between ethnic groups and different races is apotential area for expanded control of disease. The presentinvestigations showed that bulk purified mixtures of all subclasses ofhorse and rat IgG were not estrogen reversible inhibitors for MTW9/PL2rat mammary tumor cells. These results were further examined, asdescribed below.

Test of Rat. IgG Subclasses as Estrogen Reversible Inhibitors ofMTW9/PL2 Rat Mammary Tumor Cell Growth. The IgG subclasses of rat areIgG1, IgG2A, IgG2B and IgG2C. These IgGs, obtained from commercialsources previously identified herein, were tested at 15 μg/mL withMTW9/PL2 cells in DDM-2A serum-free defined medium (FIG. 119). All fourIgG subclasses were compared to rat pIgA and rat pIgM. The latter twowere estrogen reversible inhibitors, as expected (FIG. 119). However,the four IgG subclasses were not inhibitors at a concentration that waseffective with IgA or IgM. The estrogenic effects recorded in cultureswith them were no larger than seen in serum-free defined medium alone(FIG. 119). Clearly, IgG are not effective steroid hormone modulators inrat.

Test of Human IgG Subclasses as Estrogen Reversible Inhibitors of Breastand Prostate Cancer Cell Growth. The subclasses of human IgG are IgG1,IgG2, IgG 3 and IgG4. They are formed with both λ and κ light chains. Aseries of studies was performed, and it was found that with human breastcancer cells, only IgG1κ was a significant estrogen reversibleinhibitor. FIG. 120 shows a comparison of its activity to human pIgA andpIgM. At 40 μg/mL, it was 37% as effective as pIgM. A similar study withLNCaP cells showed that only IgG1κ had activity greater than theestrogenic effect seen in CAPM serum-free defined medium only (FIG.121). However, in some experiments with prostate cells, IgG2a alsoshowed androgen reversible inhibitory activity (FIG. 122). Based onthese studies, it is concluded that IgG1 and IgG2 have small butmeasurable androgen reversible activity with AR⁺ human prostate cancercells.

Discussion of Example 23. The effect of IgG1κ raises an issue notencountered with IgA or IgM. The preference for the κ light chainimplies that a different receptor mediates the effects of thisimmunoglobulin. This immunoglobulin may have greater inhibitory effectson normal breast or prostate cells that it has on ER⁺ and AR⁺ cancercells. Part of the transformation/progression process leading to hormoneresponsive cancers may be an attenuation of the effectiveness of IgG1×as an inhibitor. The present IgG1 observations have other applications,as well, including the measurement of the IgG1κ subclass in differentpopulations such as black American, Asian, white, Native American andHispanic with contrasting susceptibilities to breast and prostatecancer, or individuals within any one ethnic group, may provideadditional information and confirmation of the usefulness of suchmeasurements. These measurements can be made in bodily fluids or plasma.Measurement in milk and breast fluid may provide an indication ofsusceptibility to the development of breast cancer.

Irrespective of the receptor that mediates the growth response of IgG1κor IgG2, this receptor will be a candidate for the missing transcytosisreceptor for IgG. Its molecular identification has utility in diagnosticspecimens of breast, prostate and other cancers and can be used todetermine new uses of the immune system for therapeutic applications.Once it is completely identified, the receptor that mediates theIgG1/IgG2 growth inhibition effects will provide another target fordevelopment of compounds that mimic the immune system inhibition ofcancer cell growth. As described above with respect to the gene for thepoly-Ig receptor, the gene encoding this IgG receptor will also beuseful as a locus for analysis of genetic susceptibility to breast andprostate cancers, as well as other types of mucosal and epithelialcancers of humans.

Example 24 Mediation of IgG1κ Effects by a Fc-Like Receptor

In this, example the probable mediating receptor for IgG1κ cancer cellgrowth inhibiting effects is further described and applications forusing the gene encoding this receptor as a genetic screening tool to aidin assessing genetic susceptibility are discussed.

It is highly unlikely that IgG1 acts via the poly-Ig receptor. Thepoly-Ig receptor has a requirement for “J” chain for binding (hence itsspecificity for dimeric/polymeric IgA or pentameric IgM each of whichhas one J chain). Also, as shown in TABLE 11, Fcγ receptors arelocalized in leukocyte series or bone marrow origin cells. There is noconvincing evidence in the literature of their presence in epithelialcells or in secretory cells of the mucosa. The IgG1 inhibition-mediatingreceptor sought in the present study is one analogous to the Fcγ in twosignificant properties. First, it binds monomeric IgG1 via the Fc domainof the immunoglobulin with some participation of the κ light chain.Second, the receptor has inhibitory activity akin to a new family of Fcreceptors. The amino acid sequence of the new IgG1κ receptor is expectedto have an immunoreceptor tyrosine-based inhibitory motif (ITIM)(VxYxxL) common to a new family of inhibitory motif receptors (Cambier JC (1997) Proc Natl Acad Sci USA 94, 5993-5995). Alternatively, otheramino acid sequences may serve this same function. The Fcγ family ofreceptors contains members that possess a very special property. Theyare expected to mediate growth inhibition. The methods of identificationare outlined below.

TABLE 11 Properties of the Fcγ Family of Receptors Fcγ R1 Fcγ RII FcγRIII (CD 64) (CD 32) (CD 16) IgG1 Binding K_(a) = 10⁸ M⁻¹ K_(a) = 2 ×10⁶ M⁻¹ K_(a) = 5 × 10⁵ M⁻¹ Binding Order IgG1 > IgG1 > IgG1 = IgG3 =IgG3 = IgG3 IgG4 > IgG4 > IgG2 IgG2 Found in these MacrophagesMacrophages Natural Killer Cells Cell Types Neutrophils NeutrophilsMacrophages Eosinophils Eosinophils Neutrophils Platelets Eosinophils BCellsIt should be noted that none of these receptors has previously beenidentified in mucosal cells. Identification of one of these, or a highlyrelated growth inhibitory Fc receptor, in mucosal cells will be asignificant advance with many practical and clinical applications.

Discussion of Example 24. The amino acid sequence of a new Fc familyreceptor may include immunoreceptor tyrosine-based inhibitory motif(ITIM) common to a new family of inhibitory motif receptors (Cambier J C(1997) Proc Natl Acad Sci USA 94, 5993-5995). Fc receptors of mucosalcells that may include one of the known members of the family of ITIMs,or may contain another amino acid sequence or sequences that serve thissame function, are the subject of ongoing investigation. Once thesequence is identified, the genetic mapping to a specific chromosomenumber and locus is expected. The genomic DNA sequence of the newreceptor (or existing receptor, if already known), including introns andexons, is also expected. Once identified, this receptor will find use asa genetic screening tool for genetic susceptibility to breast andprostate and other mucosal cancers, in addition to, or analogous to,conventional breast and prostate screening technologies. Additionally,the IgG1 mediating receptor will be employed for diagnostic and clinicalapplications, as further discussed hereinbelow. Detection of mutationsand changes associated with progression from normal cells to autonomouscancer cells are using this receptor gene is foreseen. Methods ofdetecting changes in regulation or expression of the receptor due toallelic imbalances in the receptor gene are also foreseen as a new toolto determine susceptibility and prognosis for development of breast andother mucosal cancers. Detection of other regulatory and developmentalchanges are also made possible by this receptor and its gene.

Example 25 Immunoglobulin Inhibitors as Tools for Identifying theReceptors that Mediate the IgA/IgM/IgG Cell Growth Regulating Effects

This Example describes how IgA, IgM and IgG1 can serve as biologicalreagents or tools in establishing the identity of the inhibitionmediating receptors.

The Mediating Receptors—Inhibitory Function. It has been made clear bythe results presented herein, and in co-owned concurrently-filed U.S.Pat. No. ______ (Atty. Dkt. No. 1944-00201)/PCT/US2001/______ (Atty.Dkt. No. 1944-00202) entitled “Compositions and Methods forDemonstrating Secretory Immune System Regulation of Steroid HormoneResponsive Cancer Cell Growth,” hereby incorporated herein by reference,that the mediating receptor for the serum-borne agent has specialproperties. As discussed above, serum contains a great variety ofmitogenic agents. On this point the present results in 50% (v/v) serumwere especially relevant. This concentration of serum is a rich sourceof mitogens including insulin and the insulin-like growth factors.Nutrients and other serum components also have growth-promoting effects.Examples include diferric transferrin, unsaturated fatty acids bound toalbumin, complex lipids and ethanolamine. The broad range of different“mitogens” present in defined medium are described elsewhere (Riss TLand Sirbasku D A (1987) Cancer Res 47, 3776-3782; Danielpour D et al.(1988) In Vitro Cell Dev Biol 24, 42-52; Ogasawara M and Sirbasku D A(1988) In Vitro Cell Dev Biol 24, 911-920; Karey K P and Sirbasku D A(1988) Cancer Res 48, 4083-4092; Riss T L et al. (1988) In Vitro CellDev Biol 24, 1099-1106; Riss T L et al. (1988) In Vitro Cell Dev Biol25, 127-135; Riss T L and Sirbasku D A (1989) In Vitro Cell Dev Biol 25,136-142; Riss T L et al. (1986) J Tissue Culture Methods 10, 133-150;Sirbasku D A et al. (1991) Mol Cell Endocrinol 77, C47-055; Sirbasku D Aet al. (1991) Biochemistry 30, 295-304; Sirbasku D A et al. (1991)Biochemistry 30, 7466-7477; Sato H et al. (1991) In Vitro Cell Dev Biol27A, 599-602; Sirbasku D A et al. (1992) In Vitro Cell Dev Biol 28A,67-71; Sato H et al. (1992) Mol Cell Endocrinol 83, 239-251; Eby J E etal. (1992) Anal Biochem 203, 317-325; Eby J E et al. (1993) J CellPhysiol 156, 588-600; Sirbasku D A and Moreno-Cuevas J E (2000) In vitroCell Dev Biol 36, 428-446). From the present results, clearly, theimmunoglobulin inhibitor(s) also block the growth effects of all thosemitogens, and steroid hormones are selectively capable of reversing theeffects of the inhibitor(s). Plainly, as predicted by the estrocolyonehypothesis, serum contains an inhibitor that has a dominant role in theregulation of proliferation of steroid hormone target cells. Theseinhibitors will have biological implications extending well beyondestrogen and androgen target tissues. Because of its “master switch”character, the newly identified immunoglobulin inhibitors have manypractical industrial testing and manufacturing uses as well as manybeneficial clinical applications.

The Receptor Mediating IgA/IgM/IgG Inhibitory Effects. The results shownherein strongly indicate that the IgA/IgM growth inhibition is mediatedeither by the poly-Ig receptor or a very closely related receptor.Establishing a growth regulating function for this “transcytosis”receptor will open new directions in medical diagnosis, treatment andprevention of cancers of mucosal epithelial tissues. It will bedetermined whether the poly-Ig receptor, or a poly-Ig like receptor,mediates the growth regulating effects of IgA on human breast andprostate cancer cells in culture. For this study, the poly-Ig receptorin these cancer cells will be identified using well-known PCR cloningtechnology, ¹²⁵I-labeled IgA chemical cross-linking and Western andimmunohistochemistry methods that have been described in the literature.

Next, blocking polyclonal antibodies or blocking monoclonal antibodieswill be employed to show that the poly-Ig receptor mediates the growthresponse. The antibodies will be raised against the poly-Ig receptorusing known techniques. Reversal of the inhibitory effect of IgA and IgMby blocking the poly-Ig receptor will suggest that the poly-Ig receptoris not just a simple transport receptor, but that it has a central rolein breast and prostate cancer cell growth regulation. There is noexisting paradigm for breast or prostate cell growth regulation thatinvolves the poly-Ig receptor or for that matter any receptor specificfor the IgA class of immunoglobulins including Fcα receptors (Fridman WH (1991) FASEB J 5, 2684-2690).

The different forms and domains of IgG, IgA and IgM that act asinhibitors of normal prostate and breast and other mucosal epithelialcell growth and the hormone responsive and hormone autonomous forms ofthese cancers in serum-free defined culture medium will be determinedand used as tools to evidence or confirm the identity of the receptor(s)responsible for mediating the growth regulatory effect. The propertiesof the ligand that elicits a response will be evidence supporting theidentity of the receptor. Poly-Ig receptor is activated by Fc-domains asare Fcγ receptors. Normal cells are likely to be most inhibited by IgG,IgA and IgM, whereas the ER⁺ and AR⁺ cells will likely be inhibitedprimarily by IgA/IgM, and ER⁻ and AR⁻ cells will likely not be inhibitedby any of the three classes of immunoglobulins, as predicted by theconceptual model described below. The methods employed will includedirect tests of the activity of IgG, IgA and IgM on cell growth as wellas assessment of the activity of specific size forms and Fc versus Fabfragments. Antibodies such as anti-J chain and anti-Fe will be used toextend these studies to demonstrate that the Fc is the active domain andthat Fc binding receptors are involved.

More specifically, AR⁺ LNCaP cells, the AR⁻ PC3 and DU145 cells, and theAR⁺ ALVA-41 cells will be studied. Normal human prostate and breastepithelial cells will be obtained from Clonetics. Growth assays will bedone in completely serum-free CAPM (prostate) and DDM-2MF (breast), asdescribed above. IgA1 and IgA2 will be purified from human serum andcolostrum, using techniques that are well known and have been describedin the literature. Initial small samples will be obtained from acommercial supplier such as The Binding Site (San Diego, Calif.). Themonomeric, dimeric and polymeric forms of IgA will be separated usingtechniques that are well known and have been described in theliterature. If only IgA2 has activity, it will be further separated intothe A2(m)1 and A2(m)2 allotypes, using well-known techniques that havebeen described in the literature. Because the initial IgA/IgM inhibitorpreparations evaluated in the present studies were mostly dimeric andmonomeric, those forms are expected to be the most active in the futureseries of tests. Confirmation that the most active forms aredimeric/polymeric IgA/IgM will be strong evidence for poly-Ig receptormediation. Should the monomers be revealed as the only active inhibitorforms, however, it would favor Fc or Fc superfamily receptors, in whichcase the Fca will be investigated as a possible mediator.

IgA will be fragmented with a specific protease to yield Fc and Fabfragments from IgA, using techniques that are well known and have beendescribed in the literature. The Fab and Fc fragments of IgM will beobtained using a Pierce Chemicals kit based on immobilized trypsin. Faband Fc fragments of IgG1 will be obtained using another Pierce kit. Ifonly Fc fragments of IgA and IgM are active, mediation by the poly-Igreceptor is likely. If the Fc of IgG1 is active, it will indicate an Fcreceptor as the mediator.

The immunoglobulin inhibitors will also be used as tools or biologicalreagents to confirm whether IgG acts via a receptor different thanIgA/IgM. Based on the results reported above, identification of Fcγ likereceptors and the poly-Ig receptor (or related receptor) with normalcells, ER⁺ cells and AR⁺ cells is expected, and no functional receptorsare expected in ER⁻ cells or AR⁻ cells. ¹²⁵I-labeled IgG1, IgA and IgMwill be prepared using chloramine T or Iodogen® beads or coated tube(Pierce Chemicals kits). Binding parameters, binding constants, analysesof the effects of reciprocal additions of labeled and unlabeledimmunoglobulins to identify separate or similar binding sites, anddetermination of the effects of addition of purified secretory componenton IgA and IgM binding will be performed as previously described orusing well known published techniques. Specific binding will be as totalbinding minus binding in a 100-fold excess of unlabeled protein. Foreach form with activity, time, concentration and temperature dependenceof binding will be assessed. Scatchard analysis will be used to estimatethe number of sites per cell and the association constants (K_(a)).Reciprocal competitions with unlabeled and labeled immunoglobulins willbe used to define interaction with the same or different receptors. Thislatter point is important because binding of both IgA and IgM to thesame site strongly favors the poly-Ig receptor and plainlycontra-indicates Fcα (IgA) or Fcμ (IgM) receptors, which are members ofa superfamily in which each member is specific for a (monomer) class ofimmunoglobulins. In addition, the effects of blocking antibodies such asanti-secretory component, anti J chain and anti Fc will be assessed withall three cell types. Where indicated, chemical cross-linking with¹²⁵I-labeled Ig will be performed to define the mass of the receptors.Optionally, metabolic labeling and/or immunoprecipitation techniqueswill be used instead, employing well-known techniques that have beendescribed in the literature.

Western immunoblotting with normal, steroid hormone receptor positiveand steroid hormone receptor negative cell types will be performed toidentify the receptors present. Immunohistochemistry will be applied toidentify the poly-Ig receptor and Fcγ receptors on all three types ofcells using the blocking antibodies. Using a full-length human poly-Igreceptor cDNA clone, S1 nuclease protection assays will be conductedwith RNA from normal prostate and breast cells, ER⁺ and ER⁻ breastcancer cells, and AR⁺ and AR⁻ prostate cancer cells to identify mRNA. Inthe cases of ER⁺ and AR⁺ or ER⁻ or AR⁻ cells, this method will help toidentify truncated or otherwise altered receptors or non-functionalreceptors. As described in certain of the preceding examples, Westernblots have already been conducted, as well as cell growth assays withreceptor blocking antibodies. The remaining analyses will be done withnormal cells as well as all other ER⁻ or AR⁻ lines. All blockingantibodies are dialyzed against buffer containing charcoal to removeinterfering steroid hormones. Rabbit polyclonal anti secretory componentwill be raised (e.g., by HTI BioProducts, Ramona, Calif.) and rabbitpolyclonal anti-human J chain and specific antibodies against the Fcreceptors for IgG and IgA are commercially available (Accurate). Thespecificity of all antiserum will be checked by Western analysis.

To identify the receptors mediating the androgen reversible inhibitionof normal and/or AR+ cells, PCR cloning methods will additionally beused to determine the cDNA sequences of the poly-Ig receptor and Fcγreceptors from normal, AR⁺ and, if indicated, from AR⁻ cells. Thismethod will provide clear answers to the question of the relationship ofthe human poly-Ig receptor and Fcγ receptors to immune system negativeregulation. It is expected that the receptors will be found to be eitheridentical to known sequences or altered in sequence to convert them to“inhibitory motif” receptors. Based on the known cDNA sequence of thepoly-Ig receptor from HT-29 cells, PCR cloning technology will beapplied to obtain a full-length clone from the LNCaP and T47D cells.Ongoing investigations are directed to comparing receptor sequences fromnormal prostate and breast cells to identify any changes. Based on theknown sequence of the FcγRIIB1 receptor, these same studies will berepeated. The receptors identified by cloning will be examined for theimmunoreceptor tyrosine-based inhibitory motif (ITIM) amino acidsequence I/VxYxxL or related sequences. Concomitantly, the cells will beexamined by Western analysis for SHP-1 and SHP-2 phosphatase mediatorsof the inhibition of growth factor activity. These markers are not onlyassociated with the inhibitory motif but also other inhibitoryreceptors. More specifically, an LNCaP and T47D full-length poly-Igreceptor clone will be prepared and compared to the reported sequence ofthe poly-Ig receptor. The same technology will be applied to the poly-Igreceptor from normal prostate cells, and, if indicated, from the AR⁺lines. Because these cell lines are expected to express the knownpoly-Ig receptor, or a related form, the PCR approach is applicable. Thesame approach will be used with the Fcγ like receptor. However, in thiscase, because these receptors are predominantly lymphoid origin, theform in epithelial cells may be substantially different. Standardcloning methods will be employed to obtain the complete cDNA sequence ofthe Fcγ like receptor from normal and LNCaP cells. Total RNA will beextracted and mRNA purified by oligo dT cellulose chromatography (alsofor Northern analysis). cDNA synthesis will be done with oligo dTprimers and AMV reverse transcriptase followed by Rnase H to remove RNA.Second strand synthesis will be done with hexameric random primers andDNA pol. I. Treatment with T4 DNA pol, Rnase H and Rnase A creates bluntends. EcoR1 methylation is followed by EcoR1 linkers and ligation into acloning vector. (Stragene) vectors based on λgt10 (hybridizationscreening) and λgt11 (secretory component antibody screening). Bothvectors will accept inserts larger than the receptor. The cDNA sequenceof human poly-Ig receptor known is the genomic sequence. These will beused to prepare sequence specific primers for PCR. The primers willencompass the 5′ and 3′ non-coding sequences to ensure a complete cDNA.The PCR products will be subcloned using the TA kit from Invitrogen. Thesequencing of PCR clones will be done by the dideoxy chain terminationmethod (Lone Star Labs, Houston, Tex.). From these, determination ofwhether there have been significant alterations in the receptor duringthe transition from normal to ER⁻ and AR⁻ cancer cells is expected. Fromsequence data, the ITIM amino acid sequences indicating an inhibitorymotif receptor will be sought. It is important to note, however, thatthe absence of these sequences does not necessarily rule out aninhibitory function. The Western analyses for SHP-1 and SHP-2 will bevaluable as an indication of an inhibitory function even in the absenceof ITIM or when the ITIM is in a modified form.

Discussion of Example 25. Without wishing to be bound by a particulartheory, it is proposed that the inhibitory effect of IgG1 is more markedwith normal cells than with ER⁺ or AR⁺ cancer cell lines and an earlystep in the pathway to malignancy involves loss by the cell of IgG1regulation. From preliminary investigations, it appears likely that theIgA and IgM receptors are a common poly-Ig receptor (or a poly-Ig likereceptor), which in normal cells is expected to be the same as insteroid hormone receptor positive cell lines. In contrast, the IgG1receptor, likely an Fc gamma type receptor, is expected to either beeither genetically altered, or its expression altered by changes inother controls, to reduce the receptors in ER⁺ and AR⁺ cell lines. Thedemonstration that IgG1 has a major growth inhibiting effect on normalcells may lead to immunization against breast cancer by administering orenhancing IgG1 in at-risk tissues. Characterization of an inhibitoryrole for IgG1 via an Fcγ-like receptor is expected to lead to importantinnovations in medical diagnosis, treatment and prevention of cancers ofmucus epithelial tissues.

Example 26 Conceptual Model for Cascading Loss of Immunoglobulin Controlin Progression from Normal Cells to Steroid Hormone Responsive andAutonomous Cancers

Concept. The isolated inhibitors, now identified as IgA, IgM and IgG1,controlled breast and prostate cell growth by acting as a steroidhormone reversible inhibitor even when tested under the very rigorousconditions of serum-free defined culture. These active naturalinhibitors are present in blood, bodily secretions and mucosalepithelial tissues. The isolated inhibitors readily prevented the growthof these types of cancer cells when they were still in the early (i.e.,hormone responsive) stage, but not in the late, non-hormone responsivestage. These results have many implications with regard to thediagnosis, genetic screening, treatment and prevention of breast,prostate, colon and other mucosal cancers. Without wishing to be boundby a particular theory, considering the present discoveries andexperimental results and, a new conceptual model for understanding howestrogens cause ER⁺ breast cancer cell growth and for understanding howthe natural progression of breast cancers occurs to give rise to highlymalignant (and dangerous) hormone autonomous forms is proposed. Thissame model is applicable to other mucosal tissues that respond to thesteroid hormone family of hormones, including androgens and thyroidhormones.

Progression Concept based on the Breast Cancer Model—GenerallyApplicable to Mucosal Tissue Cancers. It is well established that breastcancers pass through a characteristic natural history that involves agradual evolution from near normal growth patterns into cancers that arecompletely steroid hormone autonomous (i.e. they are no longerstimulated by steroid hormones). These are usually designated estrogenreceptor negative (ER⁻). As disclosed herein, it has been found thatautonomous (ER⁻) breast cancer is accompanied by a loss in sensitivityto IgA or IgM. Fully autonomous breast cancers are not inhibited bythese secretory immunoglobulins. In light of the results describedherein, it appears that autonomous breast cancers lack the poly-Igreceptor that mediates the growth inhibiting effects of IgA and IgM.These results are of special significance because for the first timethey pinpoint a specific genetic change (i.e. in the poly-Ig receptor)that might account for the majority (i.e. approximately 75%) of breastcancers termed “sporadic” and for which there is as yet no clear geneticchange identified. Indeed, these results also provide an excellentopportunity to implement gene therapy based on reintroduction of thepoly-Ig or poly-Ig like receptor into completely autonomous cancers toregain immunological regulation.

It is well established in the literature that IgG1 is present in serumduring childhood, when breast tissue growth is precisely regulated tobody size (isometric growth). The other inhibitors, IgA and IgM, arevery low at this time, but increase in serum at puberty. Because adultwomen have increased positive stimuli for breast cell proliferation dueto estrogen production, the presence of IgA and IgM may provideadditional protection. It is now proposed that alterations in immuneregulation lead to the progression of breast and prostate cells fromnormal control to ER⁺ and AR⁺ cancer cells and that additionalalternations in immune control contribute to the development of fullyautonomous cancers, according to the following model presented in TABLE12:

TABLE 12 Model for Progression of Steroid Hormone Dependent Cancers fromNormal Growth Regulation by the Immune System to Steroid ResponsiveCancers and on to Fully Hormone Autonomous Cancers

Inhibitory Motif Receptors. The receptors mediating the immune responseregulation must be at or very near the beginning of the onset of breastcancer. Using the tools developed in the present series ofinvestigations, it is expected that inhibitory motif receptors for theseimmunoglobulins will be identified. It is now proposed that themediating receptors are members of the Ig superfamily, which includes Fcreceptors and a new class of Ig inhibitory motif receptors. This newclass of receptors has emerging importance because of the increasingrecognition of the role of negative regulation of cell growth. Thesereceptors have both common and unique properties. They bindimmunoglobulins via the Fc domains and hence can be classified as Fcreceptors. One of these is, in fact, FcγRIIB that binds IgG1 (TABLE 12)and causes inhibition of antigen activation of B cells. There are manyother examples (Cambier J C (1997) Proc Natl Acad Sci USA 94,5993-5995). Among these are more than 15 receptors now designatedSignal-Regulatory Proteins (SIRPs). These all express a specialinhibitor motif of six amino acids (I/VxYxxL) that is now referred to asthe “immunoreceptor tyrosine-based inhibitory motif” or ITIM. One of themost marked characteristics of the ITIM containing SIRPs is that thismotif recruits two phosphatases (SHP-1 and SHP-2) to result in theinhibition of all growth factor dependent proliferation. This is similarto what was observed with IgG1, IgA and IgM and ER⁺ breast cancer cellsand AR⁺ prostate cancer cells serum-free defined medium. This work isexpected to aid in the identification of the missing genes for sporadicbreast cancers and a more complete understanding of the cascade of genechanges that lead to complete loss of immune control of breast cellgrowth.

Similarly, it is suggested that alterations in immune regulation alsolead to the progression of prostate cells from normal control to AR⁺cancer cells and that additional alterations in immune controlcontribute to the development of AR⁻ fully autonomous cancers. Furtherstudies are directed at identifying a cascade of gene changes leading tocomplete loss of immune control of cell proliferation.

Similarly, it is also proposed that alterations in immune regulationalso lead to the progression of colon cancer cells from thyroid hormonereceptor (THR) normal control to THR⁺ cancer cells and that additionalalterations in immune control contribute to the development of THR⁻fully autonomous cancers. Further studies are directed at identifying acascade of gene changes leading to complete loss of immune control ofcell proliferation

Tests to determine whether steroid hormone independent breast andprostate cancer cell growth results from either the loss of the poly-Igreceptor or an inactivation of its function are a focus of continuinginvestigations. A series of steroid hormone dependent and steroidhormone independent breast and prostate cancer cell lines will becompared for their inhibitory growth responses to IgA, the presence ofpoly-Ig receptor m-RNA, the expression of the receptor by ¹²⁵I-IgAbinding analysis and immunohistochemistry localization of receptor.Detection of an absence of the receptor or an inability to bind IgA willsuggest that cancer cell autonomy arises due to a loss of secretoryimmune system regulation. Such a result would be entirely new in thefield of hormone dependent cancers and would provide a new immunemechanism responsible for conversion from hormone dependence toautonomy. New immunotherapies can be developed based on activating thereceptor in hormone responsive cancers and new gene therapies based onreestablishing the function of this receptor in autonomous breastcancers.

Ongoing investigation is directed at resolving whether hormoneautonomous breast cancer cell lines have functional poly-Ig receptors.The ER⁻ cell lines to be studied are the MDA-MB-231, BT-20, MDA-MB-330the non-tumorus BBL-100, and the Hs578t and Hs578Bst. Each will beevaluated for growth in serum-free medium±IgA and ±E₂. This study willdetermine if autonomous cells have lost immune system negativeregulation. To determine if the receptor is lost, the S1 nucleaseprotection assays will be used to seek its mRNA. A kit from AMBION willbe used. In addition, ¹²⁵I-I labeled IgA will be used to determinespecific binding characteristics as described above.Immunohistochemistry will be used to confirm and/or extend the bindingdata. If the receptor mRNA and protein are absent, these methods shouldconfirm that fact. Alternatively, if they are present but nonfunctional,these methods should also confirm that fact.

Discussion of Example 26. The proposed model of progression of mucosalcancers from normal cells to fully autonomous cancers is based on theexperimental results presented, and has not been suggested prior to thepresent invention. As previously stated, there has also been no previousrecognition of the roles of IgA, IgM and IgG1 in breast, prostate, orother mucosal cancers. The cancer progression model has diagnosticimplications. For example, breast, prostate and other cancers can beexamined for content of the IgA, IgM and IgG1 receptors, as an indicatoror aid to determining the stage of the cancer. This information can becompared to the determination of estrogen receptor and progesteronereceptor status to aid in decisions regarding immunotherapy with immunemodulators or the immunoglobulins or the use of combined anti-hormoneand immune therapy modalities. Tumors that are negative for all of theimmunoglobulin receptors are prime candidates for gene therapy toreplace the receptors and thereby reestablish immune surveillance, asfurther described in a subsequent example.

Example 27 Role of TGFβ in Breast Cancer Predicts the CellularProgression in Early Onset Breast Cancer

This Example describes a new model for TGFβ and secretory immune systemroles in cancer progression in early onset breast cancer. A “linear”progression model (e.g., normal breast cell→ER⁺ cancer cell→ER⁻ cancercell) has been generally accepted for many years (Furth J (1959) CancerRes 3, 241-265; Heppner G H (1984) Cancer Res 44, 2259-2265). Inconformity with the linear progression concept, a modified model ofhuman mucosal cell progression is presented (shown in TABLE 12) thatoutlines sequential passage of normal cells, to steroid hormonestimulated cancers that in turn give rise to steroid hormone autonomouscancers, and includes the proposed roles played by the immunoglobulininhibitors.

There exist, however, pronounced factual issues that are not adeqatelyaddressed by the linear progression model. For example, it is known thatearly onset (i.e. pre-menopausal) breast cancers are 60 to 70% ER⁻ orsteroid autonomous. This fact is difficult to explain under a strictlylinear progression model because during this time (i.e., thepre-menopausal stage) female levels of estrogen are high, and thereforeshould favor outgrowth of estrogen responsive tumors. Considering all ofthe foregoing and a number of seemingly unrelated observations, in lightof the TGFβ experimental results obtained herein, an alternative newconcept, or model, of “progression” in early onset breast cancer hasbeen reached. This proposed model is illustrated as a schematic flowdiagram in FIG. 123. This model suggests an alternative or additionalsequence for cancer progression that does not in all cases require thetransition to ER⁺ or AR⁺. As shown previously herein, TGFβ has little ifany inhibitory effect on ER⁺ breast cancer cells (FIGS. 25 and 26).However, it is also well established that TGFβ is a very potentinhibitor of normal breast epithelial cell growth (Hosobuchi M andStampfer M R (1989) In Vitro Cell Dev Biol 25, 705-713; Daniel C W etal. (1996) J Mammary Gland Biol Neoplasia 1, 331-341). Furthermore, itis equally well established that TGFβ remains an inhibitor for ER⁻autonomous cells (Arteaga C L et al. (1988) Cancer Res 48, 3898-3904;Osborne C K et al (1988) Breast Cancer Res Treat 11, 211-219). Drawingfrom the fact that ER⁺ breast cancer cells lack TGFβ receptors (ArteagaC L et al. (1988) Cancer Res 48, 3898-3904; Brattain M G et al. (1996) JMammary Gland Biol Neoplasia 1, 365-372), early onset autonomous breastcancer very likely does not arise from responsive cancer cells, butinstead arises directly from normal cells as outlined in FIG. 123 by theloss of immune surveillance. The term “immune surveillance” means thatcell growth inhibitory immunoglobulins in the general circulation,and/or secreted by or bathing the mucosal/epithelial tissues, arepresent and are in sufficient amounts to deter or prevent cancer cellproliferation. This model has many clinical implications andapplications for diagnosis and genetic screening to identify young womenat greatest risk of developing breast cancer. Early onset markers willbe loss of immune surveillance without obligatory loss of TGFβ effects.The fact that ER⁺ breast cancer cells lack TGFβ receptors while ERbreast cancer cells do have the TGFβ receptor mitigates in favor of thenew bifurcated progression model, in which both ER⁺ and ER⁻ cancersarise directly out of normal breast cells. Because it is statisticallyvery unlikely that an ER+cancer cell, after having lost the TGFβreceptor, would somehow regain that receptor before passing continuingonward to become an ER cancer cell, this non-linear alternative model isreasonable.

Discussion of Example 27. The therapeutic implications of the TGFβsystem have been reviewed (Arrick B A (1996) J Mammary Gland BiolNeoplasia 1, 391-397; Reiss M and Barcellos-Hoff M H (1997) BreastCancer Res Treat 45, 81-95). However, the model presented in the presentExample integrates the investigator's discovery of the involvement ofthe secretory immune system with the well known but complex (Koli K Mand Arteaga C L (1996) J Mammary Gland Biol Neoplasia 1, 373-380)effects of TGFβ on breast cancer cells. It is expected that a lesion inthe genetics or expression of TGFβ and/or its isoform system of threereceptors (Chakravarthy D et al. (1999) Int. J. Cancer 15, 187-194) willhave importance in modulating the estrogen reversible effects of thesecretory immunoglobulins.

Conversion of normal cells to ER⁺ responsive breast cancers involves theloss of expression of the TGFβreceptor system including one or more ofthe three different forms of the receptor. Changes in these receptors,either individually or in unison are indicated in development of steroidhormone dependent cancers. It is possible that TGFβreceptor II is ofgreatest importance of the three forms (Gobbi H et al. (1999) J NatlCancer Inst 91, 2096-2101). Nonetheless, other studies suggest receptorforms I, II and II as important. As yet, those results have not beenapplied to genetic screening related to ER⁺ breast cancers. According tothe presently proposed model, lesions in the TGFβ system precede lesionsor other types of losses of the receptors for secretory immunoglobulins.The loss of TGFβinhibitory responses may represent the earliest receptorchange identifiable in estrogen responsive breast cancer. The view thatearly onset breast cancer is a failure in immune surveillance and notnecessarily related to TGFβprovides a new focus for genetic screeningand other diagnostic tools.

Prior to the present invention, there has been no report linking theinhibitory effects of TGFβ with the inhibitory effects of the secretoryimmunoglobulins. It has been reported that TGFβ is an immune modulator(Palladino M A et al. (1990) Ann NY Acad Sci 593, 181-187; Letterio J Jand Roberts A B (1998) Annu Rev Immunol 16, 137-161). It is a member ofthe cytokine family, and as such has effects on cells of the immunesystem. It is known that TGFβ has bifunctional effects on mucosal IgAresponses (Chen S-S and Li Q (1990) Cell Immunol 128, 353-361) andinhibits IgG, IgM and IgA production by human lymphocytes (van den WallBake A W et al. (1992) Cell Immunol 144, 417-428). The discovery of thegrowth-regulating role of the immunoglobulins places the complex effectsof TGFβ in a new perspective. Increased TGFβproduction can lead tosuppression of the immunoglobulins and therefore positive growth effectson breast cancer cell growth. In the past other investigators have noteda positive effect of TGFβ on breast cancer cell growth under somecircumstances, but had no explanation for this observation (Arteaga C Let al. (1996) Breast Cancer Res Treat 38, 49-56). The results herein nowsuggest a mechanism for TGFβ positive effects on breast cancer cellgrowth. Overproduction of TGFβ is a potential issue that is pertinent tothe growth of estrogen responsive breast cancers.

Example 28 Windows of Breast Susceptibility to Carcinogenesis andMutation and the Levels of Immunoglobulin Inhibitors

In this Example, age-related changes (i.e. a reduction) inimmunoglobulin concentrations in the plasma of rats are correlated withcarcinogenesis of the mammary gland.

“Windows” and Breast Cancer. Mutations leading to breast cancer mayoccur early in life, during puberty and young adulthood, and control ofDNA synthesis by IgA/IgM during this critical period may attenuate theaction of carcinogens and reduce the risk of breast cancer later in life(Marshall E (1993) Science (Wash DC) 259, 618-621). Human female breastcancer incidence rates increase dramatically after age 50 and nowapproach one in ten by age 75. The existing data suggest that the causalmutations most likely occur at earlier ages. In view of the fact thatmilk/breast secretions decrease dramatically after menopause, it remainsto be determined whether mutations can arise later in life due to thenatural age-related reduction in the growth inhibitory function of thesecretory immune system IgA and IgM. An entirely new approach to theprevention of breast cancer is proposed, which includes administeringIgA and IgM to young female rats, initially, to diminish the effects ofcarcinogens by IgA/IgM control of DNA synthesis. These treatments arethen followed by oral “immunizations” to increase the natural levels ofimmunoglobulin secreting B-cells within the mammary tissue. This neworal immunization plan is the first attempt to prevent breast cancer bythis strategy by enhancing immune surveillance in the individual.

Entry into Phase II—in vivo Studies with Rats. The studies describedhereinabove were performed in cell culture, and constitute the Phase Istudies. That work employed well-established in vitro cell culturemodels recognized generally to yield physiologically relevantinformation. Following the in vitro studies, is Phase II, using animalmodels to further define the role of the secretory immune system inbreast cancer etiology and growth in vivo.

Mammary Carcinogenesis Literature Background. Mammary carcinogenesis infemale rodents is most effective during the developmental period thatspans early puberty through early young adulthood (Welsch C W (1985)Cancer Res 45, 341503443; Huggins C et al. (1961) Nature (Lond) 189,204-207; Janns D H and Hadaway E I (1977) Proc Am Assoc Cancer Res 18,208; Moon R C (1969) Int J Cancer 4, 312-317; Russo J and Russo I H(1978) J Natl Cancer Inst 61, 1451-1459; Dao T L (1969) Science (WashDC) 165, 810-811; Meranze D R et al. (1969) Int J Cancer 4, 480-486;Haslam S Z (1979) Int J Cancer 23, 374-379; Russo J et al. (1979) Am JPathol 96, 721-736; Gullino P M et al. (1975) J Natl Cancer Inst 54,401-414; Grubbs C J et al. (1983) J Natl Cancer Inst 70, 209-212).Single challenges with mammary specific carcinogens during this timecause tumors in the majority of animals within one year. Similarchallenges later during adulthood are far less effective. The results ofa typical carcinogen experiment with female rats are shown in FIG. 124.Those results show the effects of 3-methylcholanthrene (3MCA) anddimethylbenz[a]anthracene (DMBA). Both carcinogens are commonly used toinduce hormone responsive rat mammary tumors. Carcinogenesis is mosteffective between the ages of 30 days and 100 days, and far lesseffective in rats beyond 150 days. These data support the conclusionthat a “window” exists during which mutations can be induced that leadto breast cancer later in life. There is a strong correlation of this“window” to the timing of “terminal end bud” development in the breasttissue of female rats (Russo I H and Russo J (1978) J Natl Cancer Inst61, 1439-1449). The age relatedness of carcinogenesis in rat mammarygland is paralleled in rat ovary and rat prostate.

There is an expanding body of evidence that indicates that there is a“window” in human females in which the breast is more susceptible tocancer causing changes than at other times in life (Bhatia S et al.(1996) New Eng J Med 334, 745-793; Boice J D and Monson R R (77) New EngJ Med 59, 823-832; McGregor D H et al. (1977) J Natl Cancer Inst 59,799-811; Kaste S C et al. (1998) Cancer 82, 784-792; Boice J D (1996)Med Pediatr Oncol (Supplement 1), 29-34; Cook K L et al. (1990) AJR Am JRoentgenol 155, 39-42; Beaty O III et al. (1995) J Clin Oncol 13,603-609; Shapiro C L and Mauch P M (1992) [Editorial] J Clin Oncol 10,1662-1665). Exposure of 10 to 19 year old human females to ionizingradiation or chemical mutagens (e.g. atomic bomb survivors and patientstreated by chemotherapy and radiation for Hodgkin's disease and othercancers) leads to higher than expected breast cancer rates later inlife. Similar exposures of adult human females were far lessdeleterious. The explanation for these observations is the fact thatmammary gland DNA synthesis increases during puberty and young adulthoodis due to the onset of the differentiation program (Russo J et al.(1982) Breast Cancer Res Treat 2, 5-73) and sex hormone secretion. Asgland terminal end buds develop, they are the sites for mutagenesis(Russo J et al. (1982) Breast Cancer Res Treat 2, 5-73). Clearly, DNAsynthesis is required for carcinogenesis of mammary gland (Welsch C W(1985) Cancer Res 45, 341503443; Gullino P M et al. (1975) J Natl CancerInst 54, 401-414; Grubbs C J et al. (1983) J Natl Cancer Inst 70,209-212; Dao T L (1962) Cancer Res 22, 973-981; Dao T L and Sunderland J(1959) J Natl Cancer Inst 23, 567-581; Dao T L (1981) Banbury Report 8,281-298; Huggins C et al. (1959) J Exptl Med 109, 25-42; Nagasawa H andYanai R (1974) J Natl Cancer Inst 52, 609-610; Sinha D K and Dao T L(1980) J Natl Cancer Inst 64, 519-521; Sinha D K and Pazik J E (1981)Int J Cancer 27, 807-810). It is now proposed that this carcinogenesistiming may be due to changes in the secretory immune system negativeregulation during this critical “window” period.

Correlation of Immunoglobulin Concentrations and Carcinogenesis in RatMammary Gland. Studies were conducted to demonstrate for the first timethat the period of maximum sensitivity of the mammary gland tocarcinogenesis correlates with times of lowest IgG, IgA and IgMconcentrations in the plasma of female Sprague-Dawley (S-D)-rats.Because all three immunoglobulin classes are believed to inhibit normalmammary cell replication (TABLE 12), an antibody was selected that wouldidentify all three classes of immunoglobulins. This choice was rabbitanti-human SHBG, which recognizes the three classes of rat Ig that areof interest (FIG. 66). Before initiating these studies, two controlstudies were done to ensure that the anti-SHBG obtained from acommercial source (Accurate) effectively recognized all of the growthinhibiting activity in serum.

Immunoprecipitation of the Estrogenic Activity in CDE-horse Serum andCDE-rat Serum. The addition of various dilutions of anti-human SHBG tohorse serum effectively reduced the estrogenic activity of this serum(FIG. 125). The experiments were performed by incubation of the serumwith the designated dilution of antiserum followed by addition ofimmobilized protein A/G to absorb the rabbit antibody complexes. Eachassay started with 40% CDE-horse serum. Addition of antibodyprogressively reduced the estrogenic effect. The results in FIG. 125show that this was due to a removal of the inhibitor. A similar analysiswas repeated with CDE-rat serum from adult animals>270 days of age. Theresults are shown in FIG. 126. Anti-SHBG effectively neutralized theestrogen reversible inhibitor in serum. Additionally, the studies hereinhave demonstrated that the active fraction containing the growthregulating activity binds sex steroid hormones. To further verify thatanti-SHBG was an appropriate antibody, the experiments shown on FIG. 127were performed. The specific binding of ³H-DHT to the serum was measuredas described (Mickelson K E and Petra P H (1974) FEBS Lett 44, 34-38),followed by addition of anti-human SHBG and immunoabsorption withprotein A/G. The anti-serum neutralized the labeled steroid hormonebinding in both rat and horse serum.

Immunoglobulins in the Serum of Female Rats from Various Age Groups.FIG. 128 shows that the serum content of the immunoglobulins variedversus age, as determined by Western analysis. FIG. 128 also shows thedensitometry of the Western results with each age group. Initially at 20to 21 days of age (i.e. weaning), the Ig concentrations were at adultlevels. IgG is high immediately after weaning because of gut absorptionand placental transfer from mother's milk. Between days 34 and 60, theconcentrations of total immunoglobulins (i.e. IgG, IgA and IgM) fell by80%. Estrus begins gradually, but is active by day 41 and reaches fulladult expression by 120 days (Döhler K D and Wuttke W (1975)Endocrinology 97, 898-907; Ojeda S R et al. (1976) Endocrinology 98,630-638; Döhler K D and Wuttke W (1974) Endocrinology 94, 1003-1008). At120 days, the immunoglobulin content of the serum was againsubstantially increased. The content was even higher in multiparousretired breeders of >250 days age. Comparison of the results in FIG. 128with those in FIG. 124 indicates that immunoglobulin levels are lowestin rats when carcinogens are most effective. Notably, IgA levels inhuman females are low during childhood and early adolescence, and reachadult concentrations only after 16+ years (Leffell M S et al. (1997)Handbook of Human Immunology, CRC Press, Boca Raton, pp 86-90). Theseobservations suggest that rat and human females have the same “window”with regard to Ig including IgG, IgA and IgM. This set of facts are alsoaddressed in examples that follow.

Discussion of Example 28. This is the first study to correlate changes(i.e. a reduction) in immunoglobulin concentrations in plasma withcarcinogenesis of the mammary gland. Continuing Phase II studies willinclude an animal testing program to define the specific inhibitoryroles of IgG, IgA and IgM in mammary gland growth in vivo.

This study has additional implications. It is well known that mammarygland of multiparous females is resistant to carcinogenesis. In fact,longer-term nursing significantly reduces the risk of breast cancer. Itis also well known that the hormonal environment that accompaniesnursing establishes the secretory immune system in breast. The studiesherein lead to the concept that female hormones or other developmentalchanges increase the content of the secretory system including B cellsin breast tissue. This implies that hormone therapies must be examinedfor effects on the secretory system content of breast. This in turn canbe used to develop new agents and drugs that increase content, and hencereduce the susceptibility of breast to carcinogens or any of many otherpotential mutation causing agents or effects. Further studies aredirected at addressing this issue using carcinogen sensitive adolescentfemale rats, as well as sexually mature females and multiparous females,both of which are more carcinogen resistant than the younger females(Moon R C (1969) Int J Cancer 4, 312-317; Russo J and Russo I H (1978) JNatl Cancer Inst 61, 1451-1459; Dao T L et al. (1960) J Natl Cancer Inst25, 991-1003). The rat mammary tumor model was chosen because of thelarge carcinogenesis data base available (Welsch C W (1985) Cancer Res45, 341503443; Huggins C et al. (1961) Nature (Lond) 189, 204-207; JannsD H and Hadaway E I (1977) Proc Am Assoc Cancer Res 18, 208; Moon R C(1969) Int J Cancer 4, 312-317; Russo J and Russo I H (1978) J NatlCancer Inst 61, 1451-1459; Dao T L (1969) Science (Wash DC) 165,810-811; Meranze D R et al. (1969) Int J Cancer 4, 480-486; Haslam S Z(1979) Int J Cancer 23, 374-379; Russo J et al. (1979) Am J Pathol 96,721-736; Gullino P M et al. (1975) J Natl Cancer Inst 54, 401-414;Grubbs C J et al. (1983) J Natl Cancer Inst 70, 209-212), and theabundance of applicable methodologies. Also, there is convincingevidence that carcinogen induced rat mammary cancers are histologicallysimilar to those of human breast (Russo J and Russo I H (1978) J NatlCancer Inst 61, 1451-1459; Russo J et al. (1982) Breast Cancer Res Treat2, 5-73; Dao T L (1964) Prog Exp Tumor Res 5, 157-216; Russo J et al.(1977) J Natl Cancer Inst 59, 435-445; Murad T and vov Ham E (1972)Cancer Res 32, 1404-1415). Additionally, environmentally relevantcarcinogens (El-Bayoumy K (1992) Chemical Research Toxicology 5,585-590; Wakabayashi K et al (1992) Cancer Res Supplement 52,20922-2098s; El-Bayoumy K et al. (1995) Carcinogenesis 16, 431-434) wereselected for testing the inhibitory roles of IgG, IgA and IgM inattenuating carcinogenic effects. It is noteworthy that lipophilicpolycyclic hydrocarbons such as DMBA and 3MCA and the soluble alkylatingagent NMU effectively transform mammary tissue with single doses (WelschC W (1985) Cancer Res 45, 341503443; Huggins C et al. (1961) Nature(Lond) 189, 204-207; Janns D H and Hadaway E I (1977) Proc Am AssocCancer Res 18, 208; Moon R C (1969) Int J Cancer 4, 312-317; Russo J andRusso I H (1978) J Natl Cancer Inst 61, 1451-1459; Dao T L (1969)Science (Wash DC) 165, 810-811; Meranze D R et al. (1969) Int J Cancer4, 480-486; Haslam S Z (1979) Int J Cancer 23, 374-379; Russo J et al.(1979) Am J Pathol 96, 721-736; Gullino P M et al. (1975) J Natl CancerInst 54, 401-414; Grubbs C J et al. (1983) J Natl Cancer Inst 70,209-212) but are not found in our environment (El-Bayoumy K (1992)Chemical Research Toxicology 5, 585-590; Wakabayashi K et al (1992)Cancer Res Supplement 52, 20922-2098s; El-Bayoumy K et al. (1995)Carcinogenesis 16, 431-434). NMU has been excluded from these studiesbecause it causes specific changes in the ras proto-oncogene (Sukumar Set al. (1983) Nature (Lond) 305, 658-661; Zarbl H et al. (1985) Nature(Lond) 315, 382-385) which are not common in human breast cancers. Ithas been previously suggested that as many as 80 or 90% of human breastcancers are caused by environmental carcinogens (Higginson J (1972) In:Environment and Cancer: 24^(th) Symposium on Fundamental CancerResearch, Williams and Wilkins, Baltimore, pp 69-92; Haenszel W andKurihara M (1968) J Natl Cancer Inst 40, 43-68). To date, however, thisremains to be established.

In this series of studies, DNA synthesis will be monitored in the agegroups spanning 20 days to 270 days. When the period of maximum DNAsynthesis is identified, IgA and IgM compositions will be administered,as injections, to suppress DNA synthesis during this time. After aneffective immunoglobulin dose is found, the appropriate age group willbe treated with IgA/IgM and the effects on carcinogenesis assessedversus control animals. The expected result is that carcinogens will beless effective in those rats receiving DNA synthesis inhibiting doses ofIgA/IgM. In another series of studies, conditions for increasing B-cellpopulations in breast tissue will be determined. To begin, B cellcontent of mammary tissue will be monitored as a function of age. Thiscontrol study will then be correlated with the time period of maximumDNA synthesis. It is expected that the content of B cells will be low inthose age groups showing a maximum DNA synthesis rate. Next, using oralchallenges, it will be determined what is the most effective “immunogen”to induce an increase in B cells in mammary tissue. The end point ofthese studies will be to induce sufficient numbers of B cells to preventthe “window” increase in DNA synthesis. When conditions have beenestablished to prevent this rise, the animal will be treated withcarcinogens and monitored for tumor development and survival. This studyis expected to provide Phase II evidence supporting an oral“immunization” to reduce the effectiveness of carcinogens.

Other ongoing studies will include disruption of the function of thesecretory immune system in adult and multiparous female rats todetermine if they become more sensitive to carcinogens. Virgin femalesof 114 days or older will be studied as will breeders of more than 250days age. These animals will be treated with antibody against thepoly-Ig receptor. The doses of antiserum to disrupt the secretory immunesystem will be established by monitoring IgA/IgM secretion into bile,uterine fluids and breast milk. Also, mammary DNA synthesis will bemonitored. When secretion has been blocked effectively, susceptibilityto carcinogens will be explored. It is expected that the disruption ofthe interaction of IgA/IgM with the poly-Ig receptor will increase DNAsynthesis in the mammary gland and therefore increase susceptibility tocarcinogens. Other ongoing work will determine if mutations leading tobreast cancer occur early in life during puberty and young adulthood andwhether control of DNA synthesis by IgA/IgM during this critical periodwill attenuate the action of carcinogens and reduce the risk of breastcancer later in life.

Example 29 Risk Factors: IgA/IgM Based Test to Detect Lowered Levels ofSteroid Hormone Reversible Cell Growth Inhibitors in Plasma or BodySecretions

IgA/IgM and Cancer Susceptibility. Toward identifying individuals withhigh susceptibility to breast cancer or prostate cancer, the level ofthe inhibitory form of IgA (i.e., IgA dimer) will be measured in anindividual's plasma, or the secretory IgA and polymeric IgM will bemeasured in a bodily secretion. Decreases in plasma levels of IgA ordecreased secretory capacity into milk or structural alterations in IgAmay confer greater susceptibility to breast cancer. Levels are expectedto be low in susceptible individuals and to fall with increasing age innormal individuals, substantially mirroring the age distribution patternassociated with breast and prostate cancer incidence. One way to assayfor the dimeric/polymeric form of IgA is via a conventional antibodybinding test using antibody raised against the D5 domain disulfideregions with IgA attached. In secretory fluids, direct measure of sIgAcan be done along with a measure of secretory component byradioimmunoassay or other methods using enzyme linked immunosorbentassay (ELISA) or biotin-avidin technology, each of which are well knownin the art and have been described in the literature. The levels of IgMcan be measured directly although their levels are more subject to widevariations. Also, “J” chain can be measured, but only in samples treatedto remove the free (unbound) form known to be in plasma.

Secretory Immune System Status Test. Another informative test processwill be to use rectal or nasal passage antigen challenge and thenmeasure the appearance of the specific antibody against the antigen inplasma and secretory fluids, using standard high capacity clinical testmethods. This will directly measure the immune status of the individual.Those with optimum capacity can be separated from individuals withimpaired secretory immune system function. Impaired function of thesecretory immune system may indicate susceptibility to cancer.

Cell Growth Testing for Inhibitors. In those cases where directassessment of inhibitor in fluids is required, these can also bemeasured by cell growth assays on reduced microwell scale usingautomated colorimetric assays. The testing is carried out by firsttreating a plasma specimen to deplete or substantially remove thesteroid hormone content without inactivating or, removing the endogenouspoly IgA dimer and poly IgM molecules. The hormone depleted specimen isthen tested for cell growth inhibitory activity in the presence of addedsteroid hormone in an in vitro assay employing cultured tumor cellsincubated in a defined serum-free medium. Procedures for preparing thesteroid hormone depleted plasma or serum and for conducting the assayare described in preceding examples and in U.S. Pat. App. No. ______(Atty. Dkt. No. 1944-00201)/PCT/US2001/______ (Atty. Dkt. No.1944-00202) entitled “Compositions and Methods for DemonstratingSecretory Immune System Regulation of Steroid Hormone Responsive CancerCell Growth,” hereby incorporated herein by reference. Application ofthe XAD-4™ resin treatment is preferred for small samples. Theseextraction methods are capable of yielding steroid hormone depletedserum that allows identification of 30 to 100-fold estrogen and androgengrowth effects (cell number measurement) in culture in 7 to 14 days withhuman breast and human prostate cancer cells, as well at rat mammary,rat pituitary and Syrian hamster kidney tumor cells.

Comparison of in vitro and in vivo. The results are compared to similartests using positive and negative control plasmas or serums, which havedefined levels of IgA dimer and poly IgM. In this way the tumor cellgrowth inhibitory activity of the individual's plasma is measured.Because the in vitro assay system employs a cell line that forms breastor prostate tumors when implanted in vivo, the in vitro assay resultsare believed to be suggestive of the in vivo condition of theindividual.

Discussion of Example 29. Rats and humans process plasma and locallyproduced IgA very differently. This topic is covered in detail (Conley ME and Delacroix D L (1987) Ann Internal Medicine 106, 892-899). In rat,pIgA equilibrates with locally produced IgA and is therefore a majorsource of the immunoglobulin found in secretions. This means the IgAfrom plasma readily leaves this compartment to arrive at mucosalsurfaces and be transported by the poly-Ig receptor into the lumen ofmucosal tissues or into secretions such as bile. This physiology makesthe rat a very useful experimental tool to determine some of the cancerrelated effects of IgA and IgM. However, caution is necessary whenextrapolating rat results to humans (Conley M E and Delacroix D L (1987)Ann Internal Medicine 106, 892-899). Human plasma IgA (pIgA) does notappear to be as available to local tissues for secretion. Indeed, only asmall fraction of the secreted IgA in humans comes from plasma IgA. Thevast majority arises locally in mucosal tissues from B cells locatedthere and functioning on site. In light of this difference between ratand human IgA processing, measurement of IgA in the plasma is bestapproached from the IgA deficiency perspective described below.Measurement of the capacity of the secretory immune system in allsubjects by direct measurement in fluids (e.g. breast fluid, saliva,tears, seminal fluid, bile or vaginal washes) is preferred.

One of the best approaches to measurement of secretory immunoglobulinsin small volumes of body fluids is to challenge with an antigen to whichdifferent low molecular weight haptens are conjugated by standardchemistry now well known and very widely applied. Haptens are conjugatedto common non-antigenic proteins and identified by measuring theappearance of anti-hapten immunoglobulins in the secretory fluids. Bychanging haptens, this test can be administered many times over a periodof years.

Example 30 Risk Factors: IgA Deficiencies and Malignancies

In this Example, measurement of plasma IgA levels are correlated toincreased incidence of mucosal cancers. IgA deficiency is the mostcommon primary immunodeficiency encountered in man (Schaffer F M et al.(1991) 3, 15-44). It is very heterogeneous and is associated withinfections, allergies, autoimmune disorders, gastrointestinal diseaseand genetic disorders. An overview of immunodeficiency-associated cancerhas been presented (Beral V and Newton R (1998) J Natl Cancer InstMonograph 23, 1-6). Breast cancer risk or incidence was not consideredspecifically. Other reports have related this deficiency to abdominalT-cell non-Hodgkin's lymphoma (Ott M M et al. (1998) Am J Surg Pathol22, 500-506; Zenone T et al. (1996) J Intern Med 240, 99-102; FilipovichA H et al. (1994) Immunodeficiency 5, 91-112) and other malignancies(Pongracz K et al. (1994) Orv Hetil 135, 2863-2866). One of the mostsignificant aspects of these reports is the correlation to gastriclymphoma that is currently thought to originate from a bacterial cause.Again, breast cancer and several other mucosal cancers were either notconsidered or were discussed not considered specifically (Butler J E andOskvig R (1974) Nature (Lond) 249, 830-833). Other than the well-knownrelationship between ataxia telangiectasia with its characteristic IgAdeficiency, and breast cancer, there are no other studies of this issueknown to the inventor. This fact also extends to prostate cancers andIgA deficiencies. Measurement of plasma IgA as a measure of propensityto develop breast, prostate and other mucosal cancers is believed to beapplicable for conducting widespread screening programs.

IgM Compensation for IgA deficiency. It is of interest to note that IgAdeficiency is accompanied by a compensatory increase in IgM (BrandtzaegP et al. (1968) Science (Wash DC) 160, 789-791). Analysis of milk fromIgA deficient women indicates substantial increases in IgG subclassesand IgM (Hahn-Zoric M et al. (1997) Pediatr Allergy Immunol 8, 127-133;Thom H et al. (1994) Acata Paediatr 83, 687-691). In combined deficiencypatients, IgM levels rise sufficiently to cause IgM nephropathy (Oymak O(1997) Clin Nephrol 47, 202-203). Measurement of plasma IgA, as a toolto determine predisposition to breast cancer, can be accomplished bystandard clinical assays with high specificity antibodies to human IgA,prepared according to methods known to those skilled in the art. IgMlevels can be measured similarly.

Example 31 Risk Factors: Autoimmunity Test for Anti-IgA and IgM inPlasma

Methods and immunoglobulin inhibitors described in preceding examplesare useful for conducting studies to identify factors that are capableof neutralizing the IgA/IgM inhibitory effects on cancer cell growth.

General Applicability. IgA and IgM are estrogen reversible inhibitors ofER⁺ breast cancer cell growth in the classical sense of the long soughtafter chalones. They arrest cell growth and are readily reversed withinone week in culture and appear to be mucous epithelial cell specific infunction. These results may have implications for epithelial cancersbeyond those of breast.

Auto-Antibody Properties and Source. Anti-IgA antibodies purified fromnormal female plasma will be tested to determine if they neutralize IgAas a negative growth regulator for breast cancer cells in serum-freedefined culture, employing the cell growth assay procedures describedhereinabove. These immunoglobulins will be isolated by standard methodsin the literature and their class and subclass determined. They will befragmented to determine if activity resides in the Fab component, asexpected in view of the results described in preceding Examples.Specific blocking monoclonal antibodies will be raised against theactive component to permit measurement in the serum of females. Thepurpose of this test is to determine if an autoimmune mechanism canabrogate the negative IgA growth regulation exerted on estrogenresponsive breast cancer cells. Such studies will assist in identifyingnew factors involved in breast cancer etiology. To date, autoimmunityhas not been given significant attention with this disease. This studyis expected to reopen consideration of autoimmunity and breast cancer,and a similar approach is applicable to prostate, colon and othermucosal cancers.

Autoimmunity and Cancer. The concept that autoimmune mechanisms areinvolved in cancer development is not new. However, the present findingsshowing a direct cell growth modulating role for the secretory immunesystem is totally new. It has been reported that serum from 26 (all)normal volunteers had anti-IgA antibodies of the IgG and IgM classes(Jackson S et al. (1987) J Immunol 138, 2244-2248). They were purifiedand were directed against both polymeric and monomeric IgA1 and IgA 2containing the light chains (Fab fragments). Plasma samples will be usedto purify similar antibodies, as described above, except in this case,with the goal of isolation of Fc directed antibodies. The purifiedantibodies will be identified by class and fragmented into Fc and Fabportions. The anti-IgA antibodies will be assessed for their ability toblock the action of IgA as an ER⁺ breast cancer cell growth mediator asdescribed (Sato J D et al. (1987) Methods Enzymol 146, 63-82; Arteaga CL et al. (1988) Mol Endocrinol 2, 1064-1069; Sato J D et al. (1983) MolBiol Med 1:511-529; Gill G et al. (1984) J Biol Chem 259, 7755-7760).Those that prove effective will be used to raise specific monoclonalantibodies as described (Barret C H (1994) Hybridomas and monoclonalantibodies, In: Antibody Techniques, Malik V S & Lillehoj E P, Eds,Academic Press, San Diego, pp 71-102). After confirming by doublediffusion tests and other analyses that the monoclonal antibodyrecognizes only the appropriate anti-IgA in serum, a RIA will bedeveloped for quantification of serum samples (Lauritzen E et al. (1994)In: Antibody Techniques, Malik V S & Lillehoj E P, Eds, Academic Press,San Diego, pp 227-258). To establish a control baseline, groups of 100female serum samples will be obtained and assays done to establish abasal “normal” range for the blocking anti-IgA antibody. The age andhormonal status of the women donors will be determined. This willidentify a pattern of age differences should they occur. The effects ofestrogen containing contraceptives and estrogen replacement therapy willbe evaluated. This is especially valuable information because breastcancer occurrence is highly age dependent. Although a naturallyoccurring antibody has not yet been identified that can directly blockthe growth regulating effect of IgA, its identification will provide anew tool to measure breast cancer risk and risk for other mucosalcancers. This study makes use of several of the methods and compositionsdescribed hereinabove, including immunoglobulin inhibitors compositions,assay methods, defined media and model cell lines.

Autoimmune. Antibodies. Alternatively, or additionally, plasma andbodily fluids may be monitored for autoimmune antibodies that block theinhibitory action of IgA and IgM. An expected increase in autoimmuneantibodies with increasing age is expected to coincide with increasedcancer incidence, or the incidence of cancer may be high in individualswith early onset disease.

Example 32 Diagnostic and Prognostic Tools: Estrogen Receptor γ (ERγ)

In this Example, a new estrogen receptor is identified and its role inestrogen responsive cell growth is described. Use of the new ERγ as anadditional or replacement for ERα in gene screening procedures is alsodiscussed.

ERα as the Basis for Most ER analyses of Breast Cancer Specimens. Inpreceding Examples, a new estrogen receptor has been proposed thatregulates estrogen responsive target tumor cell growth. The measurementof this new receptor as a diagnostic and prognostic tool has greatclinical consequences. Currently, throughout the world, the measurementof the known estrogen receptor a (ERα) is accepted as the standard fordetermining whether a breast cancer is estrogen sensitive or estrogeninsensitive (Henderson I C and Patek A J (1998) Breast Cancer Res Treat52, 261-288; Osborne C K (1998) Breast Cancer Res Treat 51, 227-238;Kaufmann M (1996) Recent Results Cancer Res 140, 77-87; Allred D C etal. (1998) Mod Pathol 11, 155-168).

Candidates for the ERγ. It has been reported that a point mutation inERα causes it to become hypersensitive to estrogens (Lemieux P and FuquaS (1996) J Steroid Biochem Mol Biol 56, 87-91). The point mutation islocated in the hormone-binding domain. Growth of the human MCF-7 breastcancer cells transfected with this point mutation ERα variant isstimulated by 10⁻¹² to 10⁻¹¹M E₂ (Fuqua S A et al. (2000) Cancer Res 59,5425-5428). Those investigators proposed that this variant is a pointmutation in the ERα that occurs in premalignant breast tissue lesions.They did not suggest that it is the growth regulating form of the ERthat occurs naturally in all target cells. It should be noted that inthe preceding examples, dose-response data have been presented with manycell lines of both rodent and human origins. In every case, theconcentration that caused growth was well below the affinity constant ofthe standard ERα. This plainly raises a question about the pointmutation variant. It must be common to every cell type in thisdisclosure as evidenced by the information placed in TABLE 1, TABLE 4and TABLE 10 and the estrogen dose-response data shown in FIG. 3(MTW9/PL2 cells), FIG. 10 (T47D cells), FIG. 11 (GH₄C₁ cells), FIG. 12(H301 cells), FIG. 23B (MCF-7K, T47D and MTW9/PL2 cells), FIG. 92(MCF-7K cells) and FIG. 100 (T47D cells). To emphasize again, for thispoint mutation variant to explain all of the data herein, it must bepresent in every cell line used in this disclosure. Furthermore, theinvestigators identifying the point mutation variant made, the statementthat MCF-7 cells had to be transfected with this variant to becomesensitive to one to ten picomolar concentrations of E₂. The results ofthe studies herein show, however, that this is simply not the case withMCF-7 cells (FIG. 97) or any of the other cell lines studied. The cellsare already sensitive to one picomolar estrogen without any suchtransfection.

Search for Point Mutation Variant in the Cell Lines Used in ThisDisclosure. PCR will be used to search for the point mutation variant inthe cell lines listed in TABLE 1. This will provide a definite answer tothe question of physiological significance. Two outcomes appear mostprobable. First, the point mutation receptor is found in all of the celllines. If so, it will be cloned and transfected into ER⁻ cells todetermine if this reestablishes high sensitivity estrogenresponsiveness, as measured in the cell growth assays described inpreceding Examples. Second, if the point mutation is not found in all ofthe lines, it will indicate that the original authors were correct intheir interpretation that this variant of the ERα receptor ischaracteristic of some premalignant breast cancer lesions and not ofmore general significance. In this case, the above-describeddifferential display methods will be continued to identify the ERγ. Thegeneral domains and functions for each domain of ERα are shown in FIG.129. ERγ is expected to be homologous to ERα but to have changes in thehormone-binding domain and possibly in the transacting function and DNAbinding domains because of activation of growth related genes instead ofthe genes activated by ERα.

Applications of ERγ. The newly identified ERγ will be used inconjunction with or as a replacement for the current ERα as describedabove in the various clinical applications in use today for thediagnosis and prognostic evaluation of breast and other mucosal cancers.

Antagonists of the ERγ. The action of tamoxifen as an antagonist of theERγ will find use in the evaluation and treatment of estrogen responsivecancers. Better treatment regimes employing tamoxifen can be devisedbecause the clinician can now be better informed about the possibleeffects of the drug. Development of more effective or specificantagonists will be sought using the recombinant form of the ERγexpressed in estrogen insensitive cells and in extracts of cellsexpression the transfected receptor.

Example 33 Diagnostic, Prognostic and Treatment Decision Tools: Poly-IgReceptor (or the Poly-Ig like Receptor)

Definition of the Poly-Ig Receptor. In this Example, the poly-Igreceptor designation is intended to include the authentic poly Igreceptor as defined (Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol22, 2309-2315) or a receptor with very similar properties as describedin this disclosure. The receptor that mediates the IgA/IgM cell growthinhibitory effect is likely located on chromosome 1, as described below,although it is to some extent possible that it is located on anotherchromosome but still is a poly-Ig like receptor with the characteristicsoutlined in this disclosure.

Diagnostic, Prognostic and Treatment Mode Uses of the Poly-Ig-receptor.Breast cancer and other mucosal cancer specimens, including those fromprostate, colon, ovary, uterus, cervix, vagina, kidney and bladder, willbe assessed for the presence of ERα and/or ERγ and for the poly-Igreceptor. The cell surface receptor is preferably located and quantifiedby fluorescence immunohistochemistry after an appropriate fixation(Brandtzaeg P and Rognum T O (1984) Path Res Pract 179, 250-266) or byradioimmunoassay as described for other surface receptors (Tonik S E andSussman H H (1987) Methods Enzymol 147, 253-265). Monoclonal antibodiesagainst the whole poly-Ig receptor, the secretory component or specificdomains can also be used to quantify the receptor (Trowbridge I S et al.(1987) Methods Enzymol 147, 265-279). A variety of new enzyme-linkedimmunosorbant assays (ELISA) are also available and can be applied atvery high sensitivity based on biotin-avidin or chemiluminescencetechnology. The particular method to be applied will be dictated by thetypes of specimens supplied.

Applications of Poly-Ig Receptor Positive Results. Cancer specimensexpressing high levels of poly-Ig receptor and the ER are likely highlydifferentiated tumors for which there are treatment options. Theprognosis for these tumors is thought to be very good provided thecancer has not moved to new locations. However, metastases are adefinite negative prognostic indicator. These tumor foci can be treatedwith a combination of tamoxifen and immunotherapy either as deliveredintravenous immunoglobulins or by a natural boosting mechanism via “oralimmunization” to be discussed below. Long-term exposure to bothtamoxifen and IgA/IgM is a new non-toxic approach to treatingdisseminated cancer. Currently, disseminated breast (and other) cancersare treated by chemotherapy or possibly with radiation.

Applications of Poly-Ig Receptor Negative Results. Cancers notexpressing the poly-Ig receptor must still be assessed for ER and theprogesterone receptor. If ER positive, an appropriate treatment optionis tamoxifen or adjuvant chemotherapy. Even though tamoxifen hasactivity mimicking the immune system inhibitors, it also has activityagainst the ER (which accounts for its classification as a “mixed”antiestrogen). Immunotherapy will not be effective with these tumors.Cancers that are both poly-Ig receptor negative and ER negative areexpected to have poor prognosis. The best treatment options currentlyare limited to chemotherapy or in some cases therapy with monoclonalanti-HER/NEU. This latter treatment has proven to be of limitedapplication.

Clinical Studies of Secretory Component (Poly-Ig Receptor) Expression inColon and Breast Cancer. Others have conducted a study of the proteinand mRNA expression of the poly-Ig receptor has been done with a sampleof human colon cancers (Kraj{hacek over (c)}i P et al. (1996) Br JCancer 73, 1503-1510). In that study, expression of secretory componentwas found in 33 colorectal adenomas (31 patients) and in 19 colorectalcarcinomas from 19 patients. Although the study provides evidence thatcolon adenomas (i.e. a predisposition to colon cancer) and confirmedcancers express poly-Ig receptor, the investigators did not attempt totranslate the observations further to than to propose a role in“cellular dysplasia”.

Likewise, the levels of secretory component were measured in breasttumors from 95 patients with primary or metastatic disease (Stern J E etal. (1985) Cancer Immunol Immunother 19, 226-230). The authors of thatstudy proposed that low levels of secretory component were found inmetastatic lesions and that this “could indicate a potential forsecretory component/poly-Ig receptor involvement in immune regulation oftumor growth”. However, neither the identification of growth effectsrelated to the immunoglobulins IgA/IgM nor the identification of a roleof the poly-Ig receptor directly was investigated. That study was alsoincomplete in that there was no attempt made to determine the estrogenreceptor status of the primary or metastatic disease. Therefore, therewas no correlation to growth state based on the most accepted criterionof steroid hormone receptor status. This line of study appears to havestopped with 1985 observation. The present series of studies hasdirectly addressed the problem, however, by demonstrating growthregulation by the secretory immune system using several different ER⁺cancers. These results change the context of the diagnostic analysis ofsecretory component or poly-Ig receptor.

Example 34 Diagnostic Tools: Monoclonal Antibodies to the Poly-IgReceptor and Breast Cancer Imaging

A two-fold approach to breast cancer imaging has been devised thatincludes immunoglobulin directed and poly-Ig receptor directed methods.

Current Imaging Methods. Today, X-ray mammography remains the mostimportant method for breast cancer screening (Sabel M and Aichinger H(1966) Physics in Medicine and Biology 41, 315-368). Since the 1980s,ultrasound scanning has evolved as an indispensable adjunct to X-raymammography. Other procedures such as Doppler sonography,diaphanography, contrast enhanced MRI, CT and DSA essentially dependupon the enhanced vascularity of the tumor compared to the surroundingnormal tissue. In addition to those methods, computer assistance is usedfor signal processing which aids diagnosis by texture analysis andpattern recognition. Along with those methods, scintigraphy based onreceptors located in the breast tumors has become a new non-invasivemodality (Valkema R et al. (1996) J Cancer Res Clin Oncol 122, 513-532).The presently disclosed method depends upon expression of a specificreceptor, the poly-Ig receptor, in the breast tumor. Monoclonalantibodies to the poly-Ig receptor and/or IgA or IgM (whole molecule orfragments) will be used to image breast tumors at an early stage ofdevelopment.

Poly-Ig Receptor Directed Methods. Breast and prostate cancer cells bindpolymeric IgA and IgM, but in contrast to normal cells, the cancer cellsno longer transport the immuno globulins because of disruption of tissuearchitecture and loss of baso-lateral orientation required for secretionof the immunoglobulins. As a consequence, the immunoglobulins accumulatein the cells and are partially degraded with time. When theimmunoglobulins are radio labeled or contrast labeled, the markersaccumulate in the cancer cells compared to the amounts in thesurrounding normal cells. The cancer cells are expected to image at veryearly stages due to the accumulation of the tracer or contrast agent.Most breast and prostate cancers begin at the 1 to 2 mm tumor size, soimaging with the IgA/IgM/poly-Ig receptor should be very sensitive.Consequently, these methods constitute a significant improvement overexisting imaging systems. Many of the limitations inherent in eachimaging method outlined above will be present even with the use of IgA,IgM or poly-Ig receptor technology. Nonetheless, the knowledge baseavailable for imaging supports the use of labeled IgA/IgM/poly-Igreceptor as an improvement because the target will be very early stagetumors readily recognized by this, technology. Monoclonal antibodieswill be prepared, and radio labeled or contrast labeled IgA/IgM andreceptor will be prepared, using suitable conventional methods andtechniques that are well known to those of skill in the art.

Example 35 Diagnostic, Prognostic and Treatment Decision Tools: Fc-likeReceptor for IgG1/IgG2

In this Example, the term “Fe-like receptor” is intended to mean amember of the Fc-superfamily of immunoglobulin-like receptors, possiblywith an inhibitor ITIM motif, as described above.

Diagnostic, Prognostic and Treatment Mode Uses of the Fc-like Receptor.Of the mucosal cancers examined, evidence is presented herein forinhibitory effects of IgG on only breast and prostate cells. It islikely that IgG1 and IgG2 will have effects on other early mucosalcancers. Breast cancer and prostate cancer specimens will be assessedfor the presence of ERα and/or ERγ and for the Fc-like receptor. Thecell surface receptor is preferably located and quantified byfluorescence immunohistochemistry after an appropriate fixation(Brandtzaeg P and Rognum T O (1984) Path Res Pract 179, 250-266) or byradioimmunoassay as described for other surface receptors (Tonik S E andSussman H H (1987) Methods Enzymol 147, 253-265). Monoclonal antibodiesagainst the whole receptor or specific domains can also be used toquantify the receptor (Trowbridge I S et al. (1987) Methods Enzymol 147,265-279). A variety of new enzyme-linked immunosorbant assays (ELISA)are also available and can be applied at very high sensitivity based onbiotin-avidin or chemiluminescence technology. The method to be appliedwill be dictated by the types of specimens supplied.

Applications of Fc-like Receptor Positive Results. Cancer specimensexpressing high levels of Fe-like receptor and the ER are likelydifferentiated tumors. The prognosis for these tumors is expected to bevery good. These tumors can be treated with a tamoxifen andimmunotherapy delivered as either intravenous immunoglobulins or by anatural boosting mechanism via “oral immunization,” which is discussedin an Example that follows. Long-term exposure to both tamoxifen andIgG1κ is a new non-toxic approached to treating these cancers, asindicated by results of studies described in Examples 20 and 24,employing an in vitro model assay system that is useful as an aid forpredicting in vivo effects of a given stimulus, such as a chemical ofinterest.

Applications of Fe-like Receptor Negative Results. Cancers notexpressing the Fc-like receptor will also be assessed for ER and theprogesterone receptor. If ER positive, the preferred treatment optionsare, for example, tamoxifen or adjuvant chemotherapy. Even thoughtamoxifen has activity mimicking the immune system inhibitors, it stillhas activity against the ER (which accounts for its classification as a“mixed” antiestrogen). Immunotherapy is not expected to be effectivewith these tumors. Cancers that are both Fc-like receptor negative andER negative are expected to have poor prognosis. This diagnostic testshould indicate selection of a very aggressive chemotherapy or otherprogram.

Example 36 Diagnostic, Prognostic and Treatment Decision Tools: TGFβReceptors

In this Example, use of TGFβ in detecting early onset breast cancer andfor assessing the status of a tumor is described.

TGFβReceptors. The TGFβ receptors to be monitored will be isoforms TypeI, Type II and Type III also designated RI, RII, and RIII as described(Gobbi H et al. (1999) J Natl Cancer Inst 91, 2096-2101; Chakravarthy Det al. (1999) Int J Cancer 15, 187-194). Although breast cancers expressall three forms of TGFβ receptors, only one of these (i.e. TGFβ RIII)has been localized to a “hot spot” for breast cancer on the short arm ofchromosome 1 (i.e. 1p33-p32). Prior art studies of TGFβ expression inbreast cancer specimens have problems based on the fact that it is notclear which cell types in the tissue in fact have the receptors. Becauseclinical specimens are mixtures of cells, methods should be consideredthat establish that the target epithelial cells are either receptorpositive or negative. Immunohistochemistry of fixed tissue is thepreferred method to examine this issue. Appropriate methods have beendescribed (Gobbi H et al. (1999) J Natl Cancer Inst 91, 2096-2101).Based on that study (Gobbi H et al. (1999) J Natl Cancer Inst 91,2096-2101), the Type II receptor is most associated with breastepithelial hyperplastic lesions that increase the risk of laterdevelopment of invasive breast carcinoma. In tumor systems, Type IIreceptor is positively associated with TGFβ responsiveness (i.e. growthinhibition). As the matter stands however, the 3p22 loci for Type IITGFβ receptor (Mathew S et al. (1994) Genomics 20, 114-115) has not yetbeen mapped as a “hot spot” for breast cancer.

Diagnosis of Early Onset ER⁻ Breast Cancer. Early onset breast cancerscan be classified by measurement of their ER content, the content ofTGFβ receptors (particularly Types II and III) and the poly-Igreceptor/Fc-like receptor. Together, these assessments are expected toact as aids to define the cancer type for therapy decisions. While thesecancers are expected to be TGFβ receptor positive, therapy with this 25kDa inhibitor alone has not been effective in the past. These tumors mayrequire aggressive treatment with available tools such as standardchemotherapy or high-dose chemotherapy coupled with bone marrowtransplant.

Diagnosis of Early Onset ER⁺Breast Cancer. However, the methods outlinedabove can also be used to aid in the classification of the approximately30% of the early onset tumors that are ER⁺. These tumors are expected tobe TGFβ receptor negative. Screening for poly-Ig receptors/Fc-likereceptors plus the ERα or ERγ will indicate the use of the combinedtamoxifen (and/or newer SERMs) and immune therapy described above.Advantages of this modality are the lack of severe side effects, as wellas preservation of fertility, which is often a major consideration.

TGFβReceptors and ER⁺ Cancers. Although this discussion has been focusedon breast cancer, the same screening methods are expected to beapplicable to a number of other ER⁺ types of cancers. As shown in FIG.26, all of the ER⁺ cell lines tested appeared to be unaffected by TGFβalthough data presented throughout this disclosure shows these samelines are IgA/IgM inhibited. As can best be appreciated by referring tothe cancer progression model of FIG. 123, the combination of positiveresults with the ER (ERα or ERγ) and poly-Ig receptor (or Fc-likereceptor), along with negative results for TGFβ receptor(s) is adefining pattern for the early breast cancers that will be immune systemtreatable.

Example 37 Ataxia Telangiectasia as an Example of a Human GeneticDisorder with High Rates of Breast Cancer Coupled with an IgA Deficiency

In this Example, analogies are drawn between the characteristic IgAdeficiency in the genetic disorder ataxia telangiectasia (A-T) and therole of IgA in inhibiting steroid hormone responsive cancer growth inmucosal tissues. Homozygotes have high rates of breast cancer (Olsen J Het al. (2001) J Natl Cancer Inst 93, 121-127; Swift M (2001) J NatlCancer Inst 93, 84-85), even in males. Even heterozygotes have highbreast cancer rates (Janin N et al. (1999) Br J Cancer 80, 1042-1045;Inskip H M et al. (1999) Br J Cancer 79, 1304-1307; Lavin M (1998) BrMed J 317, 486-487; Athma P et al. (1996) Cancer Genet Cytogenet 92,130-134; Chen J. et al. (1998) Cancer Res 58, 1376-1379). The mutatedgene is thought to code a product similar to the PI-3 kinase (Savitsky Ket al. (1995) Science (Wash DC) 268, 1749-1753). However, 75% of A-Tindividuals have IgA absent or deficient. Studies have shown that theA-T lesion is not found in breast cancers (FitzGerald M J et al. (1997)Nature Genet. 15, 307-310; Bebb D G et al. (1999) Br J Cancer 80,1979-1981; Vorechovsky I et al. (1996) Cancer Res 56, 2726-2732). Thishas perplexed researchers and suggests that the high risk of breastcancer in A-T individuals may be due to factors other than the reportedgenetic lesion. Secretion of immunoglobulins by mucosal cells iscertainly impaired (Bordigoni P et al. (1982) Lancet 2(8293), 293-297;Boder E (1975) Birth Defects Orig Artic Ser 11, 255-270). Very early on,clinicians noted frequent mucosal infections in A-T individuals.

Based on the results of the studies herein, which establish the role ofIgA in mucosal/breast cell growth, it seems reasonable to suggest thatthe IgA deficiency in A-T has a direct effect on malignancy developmentin mucosal tissues, particularly breast. It is noteworthy that A-T hasbeen discussed often among breast cancer researchers as a model for theetiology of this disease, and was addressed in an editorial (Swift M(2001) J Natl Cancer Inst 93, 84-85). Tests assessing the level andactivity of IgA in an individual, according to an above-described cellgrowth assay method, can be useful for correlating to the presence ordevelopment of malignancy.

Example 38 Diagnostic and Predictive: Poly-Ig Receptor, the Fc-likeReceptor And TGFβ Receptors Based Genetic Screening for Breast, Prostateand other Mucosal Cancer Susceptibility

The mediating receptors for IgA/IgM and IgG1 inhibition, identified asdescribed in foregoing Examples, and the TGFβ receptor, will be usefulfor screening individuals for susceptibility to cancer, and for genetherapy applications to restore immune regulation in autonomous tumors.

Background Genetic Properties of the Poly-Ig Receptor. The completegenomic and cDNA sequences of the poly-Ig receptor have been determined(Kraj{hacek over (c)}i P et al. (1991) Hum Genet. 87, 642-648;Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315).Poly-Ig receptor gene has been localized to chromosome 1 at 1q31-q42locus Kraj{hacek over (c)}i P et al. (1991) Hum Genet. 87, 642-648;Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315;Kraj{hacek over (c)}i P et al. (1995) Adv ExpMed Biol 371A, 617-623).The long arm of chromosome 1 had initially been described as thelocation of the most frequent ctyogenetic abnormalities found in humanbreast carcinoma (Bieche I et al. (1995), Clin Cancer Res 1, 123-127).More recently this conclusion was modified state that distal alterationsof the short arm of chromosome 1 are the most frequent cytogeneticabnormalities in human breast carcinoma (Bieche I et al. (1999) GenesChromosomes Cancer 24, 255-263). The gene encoding the poly-Ig receptoris linked to D1S58 on the long arm of chromosome 1 (Kraj{hacek over(c)}i P et al. (1992) Hum Genet. 90, 215-219). This locus (i.e. D1S58)is a known site for “allelic imbalances” in a remarkable 75% of allbreast cancers (Loupart M-L et al. (1995) Genes Chromosomes Cancer 12,16-23). Allelic imbalances include “Allelic Loss, Allelic Gain, andImbalances”. Loss of herterozygosity (LOH) is consistently high alongthe length of the long arm of chromosome 1 at D1S58 (i.e. 46%) in breastcancers (Loupart M-L et al. (1995) Genes, Chromosomes & Cancer 12,16-23). LOH is strongly associated with development of cancer. Theobservations in this disclosure now bring meaning to this publishedobservation. The report describing changes in D1S58 did not specificwhat gene or type of gene or function might be impaired by damage tothis locus (Loupart M-L et al. (1995) Genes, Chromosomes & Cancer 12,16-23). The Inventor's results indicate this “hot spot” is either theauthentic poly-Ig receptor acting in its new capacity as a growthregulator, or a very closely related receptor with similar molecularweight, ligand binding and immunological properties. However, it must berecognized that the functional form of the growth regulatory receptormay arise from alternate splicing of the poly-Ig receptor gene.Alternate splicing of the poly-Ig receptor gene is known in rabbit(Deitcher D L and Mostov K E (1986) Mol Cell Biol 6, 2712-2715; FrutigerS (1987) J Biol Chem 262, 1712-1715) and bovine tissue (Kulseth M A etal. (1995) DNA Cell Biol 14, 251-256). It has yet to be proven (ordisproved) in humans. Certainly this possibility is still open withhormone responsive cancer cells. Alternately the 1q3′-q41 region ofchromosome 1 contains several other genes of immunological interest(Kraj{hacek over (c)}i P et al. (1991) Hum Genet. 87, 642-648;Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315; BrunsG A P and Sherman S L (1989) Cytogenet Cell Genet. 51, 67-77). As shownin FIG. 130, the locus of the poly-Ig receptor (PIGR) is distant fromthe major other loci for breast cancer locatedcon chromosome 1. TheEntre Genome NCBI Search listed 31 “hot spots” for mutations occurringin breast cancer specimens. None of these genes were related to thepoly-Ig receptor. An expanded diagram of chromosome 1 is shown in FIG.131. It further emphasizes the fact that the locus of the poly-Igreceptor will represent a new discovery as a breast cancer gene. Therecan be little doubt that the discovery herein of immune negativeregulation of growth mediated by the poly-Ig receptor, or one veryrelated, is an advance. It was arrived at not by the genetic approachdescribed above which screens genes without regard for function, butinstead by a functional approach based on the biochemical, endocrine andcell biology studies described above.

Identification of the Poly-Ig Receptor by cDNA Sequencing. Molecularcloning and cDNA sequencing of the IgA/IgM inhibition mediating receptorhas been generally described in a preceding Example. Preferred ways ofcarrying out those procedures for identifying the poly-Ig receptor areprovided next. The complete cDNA sequence of the poly-Ig receptor willbe established by PCR cloning or cDNA cloning with antibody screening asdescribed in TABLE 13. This will be done with ER⁺ T47D human breastcancer cell lines and the LNCaP prostate cancer cell line. These lineswere chosen because they express either the authentic poly-Ig receptoror one very similar as determined by antibody blocking activity (FIGS.114 and 115). Also, by Western analysis the LNCaPcells express ananti-secretory cross-reacting band of the same molecular weight asauthentic poly-Ig receptor from HT-29 cells (FIG. 116). This sametechnology will be used to obtain the Fc-like receptor from the same twohuman cancer cell lines because they were shown to be responsive toIgG1/IgG2 (FIGS. 120, 121, and 122). Two strategies appear useful forthis procedure and summarized in (TABLE 13). The selection of theappropriate strategy depends upon the results of the studies outlinedabove. For example, if a poly-Ig like or an Fc-like receptor is sought,there is sufficient sequence data available to apply PCR cloning. If anentirely new receptor is expected from the receptor biochemistrystudies, cDNA cloning will be required with antibody screening. PCRcloning will be done according to published detailed procedures (CurrentProtocols in Molecular Biology, Volume 3, (2000) Sections 15.6 & 15.7,cDNA Amplification Using One Sided (Anchored) PCR and Molecular Cloningof PCR Products). The cDNA cloning will be done with the Lambda TriplEx®Phagemid which gives a three fold greater chance of finding positiveplaques with antibody. The complete manual for cloning and use of thisvector has been obtained by Internet from ClonTech, January 1996CLONTECHNIQUES.

TABLE 13 Molecular Cloning Strategies for the Poly-Ig Receptor and theFc-like Receptor STRATEGY 2: cDNA CLONING/ STRATEGY 1: PCR CLONINGANTIBODY SCREENING 1. Prepare poly (A)⁺ RNA 1. Identify inhibitoryreceptor blocking antibody 2. Use oligo (dT) primer and RT to 2. Preparepoly (A)⁺ RNA make cDNA 3. Use cDNA with specific primer 3. Use oligo(dT) primer and RT plus oligo (dT) primer to amplify to make cDNA withTaq DNA Polymerase 4. Amplify again with cDNA and 4. Methylate, makedouble internal primers plus oligo (dT) with stranded DNA Taq DNAPolymerase 5. Check size by agarose gels 5. Select Vector (TriplEx in akit) (single band) 6. Clone into AT vector for DNA 6. Ligate DNA intovector sequencing 7. Primers selected with on-line 7. Introduce vectorinto E. coli computer assistance 8. Screen with blocking antibody 9.Amplify clones for DNA sequencing 10. Because the 5′-end may be missing,use Rapid Amplification of cDNA ends (5′-RACE) kit (Ambion) to get afull length clone.

Receptor Identification and Chromosome Localization. The molecularcloning of both receptors will provide structural identification anddetermine if the poly-Ig receptor is the authentic form previouslyassociated with only transcytosis, or whether it is an altered form. Thesequencing results are expected to resolve the alternate splicing issuediscussed above. If the sequence results indicate a new receptor,chromosomal localization will be done to determine if it is within theD1S58 linked locus on chromosome 1 or possibly on another chromosome. Ifit is located on another chromosome, this will be solid evidence of anew Ig superfamily receptor gene that negatively regulates growth. Thesame discussion applies to the Fc-like receptor. It is expected that theFc-like receptor will be a new gene because of the data showinglocalization of the other known forms to leukocyte series cells (TABLE11). Additionally, the amino acid sequences deduced will be used tomatch to known ITIMs to determine whether the inhibition regulatingreceptors are members of this new class of inhibitory receptors, asdiscussed above.

Transfection Studies to Regain Immune Regulation and Steroid HormoneResponsiveness. One ER⁻ cell line will be selected for transfectionbased on Western analysis demonstrating a lack of receptor expression.Also, the DU145 cells and ALVA-41 human prostatic carcinoma cells willbe used. These cell lines are AR⁺ but are not inhibited byimmunoglobulins (FIG. 117). Transfection of these cells is expected torestore IgA/IgM inhibition and possibly permit demonstration of androgenreversibility. If this is identified, it is very strong evidence for thepositive/negative model proposed herein as the control mechanism forsteroid hormone sensitive cells. For the transfection studies, receptorcDNA will be subcloned into a mammalian cell expression vector. A vectorwith a CMV promoter will be used because of its wide range of tissueexpression and high levels of product. This will include a six aminoacid sequence of c-myc oncogene to detect transformants. This tag willallow the laboratory to distinguish between low levels of endogenousexpression and expression due to transformation. The transfections willbe done with cationic detergents. This protocol will use the GreenFlourescent Protein (GFP) reporter (CMV promoter) which can bevisualized directly without fixation or staining. Transient expressionof the receptor will be monitored for 80 hours by c-myc immunodetection.To measure the growth inhibitory effects of the IgA or IgM during thistime, tritium labeled thymidine incorporation into DNA will be measured.For longer-term studies, stability-transformed cells will be selectedusing the antibiotic neomycin and G418. Stable transfectants will bemonitored for receptor expression as described above. If stabletransfectants regain immune control, this will be reasonable support forthe conclusion that an effective receptor has been identified. This isan important precursor study for the use of the receptors in genetherapy of breast and prostate cancers a well as other mucosal cancers.

Site Directed Mutagenesis to Identify Critical Domains. Transfectionwith the tissue culture models above will be used to identify and/orconfirm domains in which mutations cause loss of the receptor function.This is an important control because all genes have variations that mayor may not be critical. This has certainly been true of BRCA1 (Iau P Tet al. (2001) Eur J Cancer 37, 300-321). Standard site directedmutagenesis methods are planned to alter specific amino acids or partsor all of selected domains. These cell culture studies will be matchedto the sequences being derived from non-disease females to definenatural variations that have no effect versus changes that aresignificant. In the case of BRCA1, the presence of a specific mutationin families with breast/ovarian cancer was used as an importantindication of changes that were significant (Iau P T et al. (2001) Eur JCancer 37, 300-321).

Predictive Genetic Analysis: Germ Line Mutations. Women with familyhistories of breast cancer especially in first-degree relatives arecandidates for genetic analysis of the poly-Ig receptor and/or theFc-like receptor. These analyses will rely on the knowledge of theimportant domain or other mutations that have been defined by monitoringwomen with breast cancer versus those without disease as well asinformation gained above by site directed mutagenesis. The availabilityof a direct biological assay of receptor function versus mutationposition and/or type is a distinct advantage over the situation withBRCA1 and BRCA2 (Iau P T et al. (2001) Eur J Cancer 37, 300-321). Themethodology is well described (Malkin D et al. (1990) Science (Wash DC)250, 1233-1238). Skin bioposy fibroblasts or blood leukocytes areextracted to obtain DNA. Using PCR, selected exons will be amplified andDNA sequenced. Multiple primers can be used to cover the whole receptor,especially if it is similar to the eleven-exon structure of the poly-Igreceptor. Generally, Fc-like receptors are >70 kDa, indicating evenfewer exons. Both DNA strands will be amplified. As technology develops,the traditional slab-gel electrophoresis analysis will preferably bereplaced with high throughput mutation screening using automatedcapillary electrophoresis (Larsen L A et al (2000) Comb Chem HighThroughput Screen. 3, 393-409). This will facilitate commercialscreening of large numbers of DNA samples. A significant mutation in oneallele is a potential predisposing factor based on the need for only oneadditional “hit” to have a loss of a critical receptor. These samechanges may be applicable to prostate, colon and other mucosal cancers.

Predictive Genetic Analysis: Other Allelic Imbalances. There are avariety of other potential genetic changes that may predispose women tobreast cancer. Changes that are especially relevant to this disclosureinclude loss of heterozygosity (LOH), concomitant gain and loss ofalleles (GAL) and simple gain of alleles (GCN) (Loupart M-L et al.(1995) Genes Chromosomes Cancer 12, 16-23). The effect of each of theseis to increase genetic instability and contribute to changes that affectthe expression of the gene product. These will be further addressedbelow in Examples related to tumor diagnostics. These same changes maybe applicable to prostate, colon and other mucosal cancers.

Predictive Genetic Analysis: Expression Genetics in Cancer. One of themost interesting facts of cancer is that relatively few have beendirectly related to mutated genes in humans (Sager R (1997) Proc NatlAcad Sci USA 94, 952-955). What is far more common is that expression ofgenes is changed. The definitions of the different types of changes are“Class I genes” that are mutated or deleted at the DNA level, and “ClassII genes” that are not altered at the DNA level but are changed inexpression level. In this disclosure, both types of changes are includedfor the poly-Ig receptor (or poly-Ig-like receptor) and the Fc-likereceptor. The information gained from characterizing these changes willbe used to improve the diagnosis, prognosis, treatment or prevention ofmucosal cancers.

TGFβ Receptors and Genetic Analysis. The protocols just described abovefor application to the poly-Ig receptor and the Fc-like receptor arealso applicable to, and are hereby extended to include, the TGFβ Type I,Type II and Type III receptors with breast and prostate cancer,preferably. It can readily appreciated that similar analyses can beapplied to other mucosal cancers as they are proven to be regulated byIgA/IgM. The genetic analysis of Class I and Class II changes in TGFβreceptors will preferably be done in combination with evaluations of thestatus of ERα and/or ERγ and the immunoglobulin receptors, as an aid inselection of the most appropriate therapy for a particular patient.

Primary Tumor Analysis. Primary tumors will be screened for allelicimbalances as described (Loupart M-L et al. (1995) Genes ChromosomesCancear 12, 16-23). Based on the known allelic imbalances associatedwith breast cancer and locus D1S58, these will be preferred analyses.Other analyses such as chromosomal loss and chromosomal rearrangementsare recognized as important aspects of cancer development andprogression (Lengauer C et al. (1997) Nature (Lond) 386, 623-627) andwill be included as receptor identification loci are defined.

Molecular Assessment of Cancer. There are several major advances incancer genetics arising from the present invention that promise a newclinical future for cancer diagnosis, genetic screening, prevention andtherapy. These include: (1) A detailed definition of the genetic (DNA)changes and altered gene expression will become available for mucosalcancers and will include the new receptors disclosed herein. (2)Obtaining the genetic profile of a single patient's primary tumor willbecome a routine matter and permit far better design of treatment formucosal cancers. (3) Large scale population based screening will becomea reality with samples obtained by non-invasive procedures or fromeasily assessable body fluids such as saliva, sputum, urine and mucosalwashings. Representative applications of these concepts and approachesare described herein. (4) A molecular analysis of surgical margins andlymph nodes and metastases will become routine, particularly for mucosalcancers, as evidenced herein. (5) The information provided in thepresent disclosure, and the tools and methods developed and describedherein will be of especial value when applied to the preinvasive andpreneoplastic states of mucosal cancers before they become symptomatic.

Example 39 Breast Cancer Prevention with Applications to Prostate Cancerand Other Mucosal Cancers

Oral immunization strategies have been devised to reduce the risk ofand/or prevent breast cancer and cancers of other mucosal tissues.

World-Wide Breast Cancer Death Rate by Country. When expressed by deathrate per 100,000 population, it is clear that the ranking (1 highest and44 lowest) is highest in industrial/developed countries of North Americaand Northern. Europe (TABLE 14). Large Asian populations are at thebottom of the ranking. The conventional wisdom is that the populationsof high-ranking areas are exposed to more environmental carcinogens andmutagens, and also have the highest dietary caloric and fat intake. Thishas led to the general acceptance of the idea that diet and environmentcause breast cancer.

TABLE 14 World-Wide Death Rates for Breast Cancer Deaths per 100,000COUNTRY/RANK Denmark/1 Ireland/2 Netherlands/3 Israel/4 United Kingdom/5Hungary/6 New Zealand/7 Germany/8 Trinidad & Tobago/9 Canada/10Solvenia/11 Czech Republic/12 Austria/13 United States/14 Australia/15France/16 Norway/17 Lithuania/18 Estronia/19 Croatia/20 Republic ofMoldova/21 Portugal/22 Spain/23 Latvia/24 Finland/25 Sweden/26 Greece/27Russian Federation/28 Poland/29 Macedonia/30 Bulgaria/31 Romania/32Cuba/33 Kazakhstan/34 Chile/35 Venezuela/36 Kyrgyzstan/37Turkmenistan/38 Mexico/39 Columbia/40 Mauritius/41 Azerbaijan/42Japan/43 China/44 Slovakia - no data

Comparisons of the World-wide Death Rates for Colon/Rectal, Breast andProstate Cancer. Of the major mucosal cancers, colon/rectal, breast andprostate are the most common and have high mortality in many countries.FIG. 132 shows a listing from the World Health Organization (1999) ofthe deaths per 100,000 in 45 countries. Although the correlations arenot ideal, the general conclusion is that several of the high rankedcountries have above average rates of all three types of cancer. Thesecountries again tend to be the industrialize/developed with the dietaryand environmental problems associated with higher standards of living.These statistics show that mucosal cancer is a common problem in moreaffluent countries and that prevention is a major problem that hassignificance in broad areas of the world.

Plasma Immunoglobulins and Age in Humans. It is well recognized thatduring the first few months of life, the immune system of the infant hasnot yet developed. The immunoglobulins in the child's blood are from themother and are predominantly IgG subclasses IgG1 and IgG2 (FIG. 133). Asshown in FIG. 133, IgM is the next Ig to increase as early as in thefirst year. This rise is required for the development of the full immuneresponse. Notably, IgA is much slower to reach adult levels and onlyachieves this after age 10+. The late appearance of plasma IgA isparalleled in some of the mucosal tissues. Reproductive system mucosalimmunity of males and females is hormone dependent and does not developuntil the onset of puberty, and then only reaches adult levels wellafter this time. This indicates that during the period of development ofthe breast adolescent females, the secretory immune system is justdeveloping. This is the “window” of opportunity for mutation describedabove. If this window were reduced, or its open period decreased, asignificant reduction in breast cancer risk could be expected.

Prevention of Breast and Prostate and other Mucosal Cancers by “OralImmunization”. Development of a broadly applicable immunization approachto prevent mucosal cancers is urgent. Today, there is no suchimmunization method. In the present Example, the observations and datapresented above establishing the inhibitory effects of the secretoryimmune system are extended to the development of an oral immunizationmethod based on induction of increased immunoglobulins in mucosaltissues. This increase is expected to slow DNA synthesis and therebyreduce the effect of mutagens during the adolescent female “window”.Furthermore, there is another “window” caused by menopause. At thistime, the secretory immune system of breast decreases. This reducesavailable inhibitors. Existing preneoplastic cells are no longer undersufficient negative control. It is proposed that this natural process isa major contributor to the sharp rise in breast cancer incidence aftermenopause.

Stimulation of the Body's Natural Immune System to Close “Windows”Periods of Mutagen Susceptibility—Dual Benefits. Breast cancer will beused as a model of mucosal tissues, employing a new approach topreventing or reducing the risk of breast/prostate/mucosal cancer bystimulating the body's natural mucosal immune defense system, preferablyvia oral immunogens, to prevent early mutations that ultimately lead tocancer later in life. Evidence presented herein shows that longer-termexposure of ER⁺ breast cancer cells to IgA or IgM will result in celldeath within a few weeks in culture. Even given that this process willtake longer in vivo, use of oral immunization throughout adult lifepromises benefits. By approaching oral immunization from thisperspective, it becomes both prevention and therapy.

Gastrointestinal Immune System. It is now proposed that “oralimmunization” can be administered to men and women of all ages tostimulate the natural secretory immune system to produce increased localtissue antibacterial immunoglobulins IgA and IgM (Del Giudice G et al.(1999) Immunol Methods 19, 148-155). Because of their well establishnatural antimicrobial properties (Heremans J F (1970) In:Immunoglobulins, Biological Aspects and Clinical Uses, Merler E ed.National Academy of Sciences, Washington, D.C.), secretoryimmunoglobulins can be expected to prevent or substantially reduce therisk of breast and prostate cancer. The presently disclosed methods andcompositions, directed toward prevention, promise to be applicable toreducing the risk of breast cancer in women without regard to age, race,existing risk factors, ethnic background or socio-economic status. Thisis true also of the risk of prostate cancer in men.

B Cells and Peyer's Patches. B cells of the lamina propria secrete IgAand IgM in breast and prostate tissue. These cells originate from thePeyer's patches of the small intestine (Owen R L (1999) Seminars Immunol11, 157-163) and migrate to breast and prostate after a maturationprocess in the circulation. B cells from the gut enter the generalcirculation after stimulation by oral agents (Boyaka P N et al. (1999)Am J Trop Med Hyg 60 (4 suppl), 35-45). This includes bacterial andviral challenge. The IgA and IgM produced in breast tissue is secretedinto milk (Nathavitharana K A et al. (1995) Arch Dis Chil Fetal NeonatalEd 72, F102-F106). The IgA and IgM produced in prostate tissue issecreted into seminal fluid (Stern J E et al. (1992) J Reprod Immunol22, 73-85). The immunoglobulins are transported across mucosalepithelium by poly-Ig receptor mediated transcytosis (Mostov K E (1994)Annu Rev Immunol 12, 63-84). In all secretions of mucosal tissues, IgAand IgM are primary antimicrobial agents. This process has beendescribed in detail (Mestecky J and McGhee J R (1987) Adv Immunol 40,153-245). After identifying the types and strains of bacteria mostlikely to cause breast and prostate cancer, the researcher proposes touse inactivated forms or attenuated forms as oral challenges to developmucosal immunity (Viret I F et al. (1999) Infect Immunol 67, 3680-3685).As evidence of the feasibility of this concept, this same approach wasused by Sabin to develop mucosal immunity against the poliovirus(Valtanen S et al. (2000) J Infect Dis 182, 1-5; Fiore L et al. (1997) JVirol 71, 6905-6912).

Oral Immunization. Oral immunization can be effective for induction ofspecific sIgA responses if the antigens are presented to the T and Blymphocytes and accessory cells contained within the Peyer's patcheswhere preferential IgA B-cell development is initiated. The Peyer'spatches contain helper T cells (TH) that mediate B-cell isotypeswitching directly from IgM cells to IgA B cells then migrate to themesenteric lymph nodes and undergo differentiation, enter the thoracicduct, then the general circulation, and subsequently seed all of thesecretory tissues of the body, including the lamina propria of the gutand respiratory tract. IgA is then produced by the mature plasma cells,complexed with membrane-bound secretory component, and transported ontothe mucosal surface where it is available to interact with invadingpathogens. The existence of this common mucosal immune system explainsin part the potential of live oral vaccines and oral immunization forprotection against pathogenic organisms that initiate infection by firstinteracting with mucosal surfaces.

Oral Immunization is Not Conventional Tumor Immunization. In view of theforegoing examples, it can be readily appreciated that a primary goal inthe present case is not to raise conventional anti-tumor antibodiesagainst the tumor, in contrast to existing approaches commonly usedtoday for cancer immunotherapy. Available mucosal routes for obtainingthe desired immune response (i.e., production of IgA/IgM/IgG1) includeoral, intragastric, nasal, urogenital and rectal. Oral administration ispreferred, however, because of its ease of use, whether for inducingmucosal secretion of cancer-arresting amounts of IgA/IgM/IgG1 in contactwith the gastrointestinal mucosa or at another mucosal site. Nasaladministration can be effective and convenient.

Strategies for Immunization A number of suitable strategies have beendeveloped for oral immunization, including the use of attenuatedmutants, of bacteria (e.g. Salmonella) as carriers of heterologousantigens, encapsulation of antigens into microspheres composed ofpoly-DL-lactide-glycolide (PGL), protein-like polymers-proteinoids,gelatin capsules, different formulations of liposomes, adsorption ontonanoparticles, use of lipophilic immune stimulating complexes, andaddition of bacterial products with known adjuvant properties, all ofwhich are well known to those of skill in the art and have beendescribed in the literature.

Age to Begin Oral Immunization. Prevention is one of the most importantissues in cancer. It is well known that there is a time period, orwindow, during which young females are most susceptible to mutagenicevents (e.g., ionizing radiation and/or exposure to chemical mutagens)that later predispose them to higher than average rates of breastcancer. This window is during puberty (i.e. about 9 to 16 years). Anoral “vaccine” will be given to very young females (i.e. starting asearly as seven years of age, or less) to induce high levels of tissue Bcells that secrete protective dimeric IgA and pentameric IgM. This sameprotective treatment or preventative may also be administered to womenof all ages, with the goal is to “immunize” women against breast cancerby increasing the tissue concentrations and secretion of polymeric IgAand IgM. This very same process can be applied to prostate and manyother types of epithelial tissues and cancers.

Rising Risk of Breast Cancer. The risk of developing breast cancer forwomen in the United States has been rising steadily for the past severaldecades. It will soon approach one in eight. We are fortunate that newtreatments and more effective screening tests have kept mortality ratesfrom also rising as dramatically. Nonetheless, we are still losing morethan one hundred women per day to breast cancer in the United Statesalone. It is generally recognized by breast cancer researchers that thefirst line of defense against this disease is prevention. In the nearfuture, the present know-how will continue to be used to treat thesecancers as they occur. However, in order to improve the long-termoutlook for all women, and especially if we wish our daughters to livefree of this disease, major efforts must also be focused on prevention.

The Secretory Immune System and Growth Regulation as the DiscoveryOpening this New Area of Prevention. As detailed in the precedingexamples, a major breakthrough has been made in understanding how breastcancers grow. It was, found that in its initial stages, breast cancer isinhibited by the secretory immune system. That means this part of ourimmune system can stop early cancer cells from growing. The well knownoperation of the secretory immune system includes, during adult life,the production by women's breasts of milk or milk-like fluids. Milkcontains high levels of two immunoglobulins IgA and IgM. These arepassed from mother to child during breastfeeding. Both IgA and IgMprotect the child's digestive system from bacterial infections. Alongwith protecting the child, we have known for many years thatbreastfeeding lowers the risk of breast cancer. As a result of thisdiscovery, it is now recognized that the same immunoglobulins thatprotect a child from bacteria can also be manipulated to protect themother against breast cancer. This realization also provides new insightwith respect to the problem of prevention.

Oral Immunization—Mass Applicability. If the secretory immune system canbe stimulated at times when women are known to be most susceptible toenvironmental and other agents that cause breast cancer, the occurrenceof breast cancer might be prevented or at least the risk of developingthis disease might be considerably reduced. Although there have beenprevious studies in the literature relating to cancer prevention, noneof the studies contemplating the use of oral immunization to treatcancer or a wide variety of infectious diseases, had pursued thatobjective beyond initial thoughts. Moreover, the application of oralimmunization specifically to breast cancer had not received anyattention. One benefit of the new oral immunization strategy forreducing the risk of and/or preventing breast cancer is that oralimmunization is readily adaptable to mass populations of women of allages and all circumstances throughout the world.

Example 40 Rat Model for Testing Oral Immunization Effects on MammaryGland Carcinogenesis

Rat Mammary Tumor Model For “Windows.” In this Example, use of an animalmodel to test the effectiveness of oral immunization during specificwindows of susceptibility to carcinogens is described. This study isintended to be conducted before advancing to any type of human testing.Mammary carcinogenesis in female rodents is most effective during thedevelopmental period that spans early puberty through early youngadulthood (FIG. 123). Single challenges with mammary specificcarcinogens during this “window” period cause tumors in the majority ofanimals within one year. Similar challenges later during adulthood arefar less effective. The results of two typical carcinogen experimentsare shown in FIG. 123. These data support the conclusion that a “window”of increased susceptibility exists during which mutations can be inducedthat lead to breast cancer later in life. There is a body of evidencethat indicates that this is also true of human females. Exposure of 10to 19 year old females to ionizing radiation or chemical mutagens leadsto higher than expected breast cancer rates later in life. Similarexposures of adult human females were far less deleterious. Theexplanation for these observations is the fact that mammary gland DNAsynthesis increases during puberty and young adulthood due to the onsetof the differentiation program and sex hormone secretion. This programinitiates the full development of the gland. As gland terminal end buds(TEB) develop, they are the sites for mutagenesis. Clearly, DNAsynthesis is required for carcinogenesis of mammary gland. Takingadvantage of these facts, it is proposed that the secretory immunesystem can be stimulated to reduce DNA synthesis during this critical“window” and thereby diminish the risk of carcinogen induced breastcancers. In FIG. 128, it was demonstrated that IgA in the plasma offemale S-D-rats is significantly reduced at the time when carcinogenesisis most effective.

Carcinogen sensitive adolescent female rats as well as sexually maturefemales and multiparous females, both of which are more carcinogenresistant than the younger females will be studied. The rat mammarytumor is a suitable model because of the large carcinogenesis databaseavailable and the abundance of other applicable methodologies. Also,there is convincing evidence that carcinogen induced rat mammary cancersare histologically similar to those of human breast. Preferably,environmentally relevant carcinogens will be employed in the studies.While lipophilic polycyclic hydrocarbons such as7,12-dimethylbenz(a)anthracene (DMSA) and 3-methylcholanthrene (3MCA)and the soluble alkylating agent nitrosomethylurea (NMU) effectivelytransform mammary tissue with single doses, they are not found in ourenvironment. NMU is also excluded from these studies because it causesspecific changes in the ras protooncogene that are not common in humanbreast cancers. Investigators have suggested that 80 to 90% of humanbreast cancers are likely induced by environmental carcinogens.

Inhibitory compositions containing IgA, IgM and/or IgG1 will be employedto determine whether mutations leading to breast cancer occur early inlife during puberty and young adulthood, and if the control of DNAsynthesis by IgA/IgM during this critical period will attenuate theaction of carcinogens and thereby reduce the risk of breast cancer laterin life. IgA and IgM will be administered to young female animalsinitially to diminish the effects of carcinogens. These studies willthen be followed by oral “immunizations” to increase the natural levelsof immunoglobulin secreting B-cells within the mammary tissue. Thestudies will include adolescent females as well as those in mid-life. Intreated individuals, there may be some consequential delay of entry intopuberty and/or some reduction in breast development, compared tountreated individuals. This oral immunization approach is the firstattempt to deter or prevent breast cancer using the new strategy, and isfurther unprecedented by applying it early in life.

General Materials and Methods. S-D female rats will be purchased fromHarlan-Sprague-Dawley. Animal holding rooms are maintained at 23±2° C.at constant humidity on 12 hour light/12 hour dark cycles. Afteranesthesia, blood will be drawn by cardiac puncture untilexsanguination. The blood will be clotted overnight at 4° C. beforecollection of serum. The serum from individual animals will be storedseparately at −20° C. The rats will be fed an AIN-76A high fat dietwhich was effective in another study of mammary carcinogenesis with S-Drats treated by gavage with the environmentally ubiquitous agentsbenzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and2-amino-1-methyl-6-phenylmidazol[4,5-b]pyridine (PhiP). This dietsupports body weight gain at control levels even after eight weeklycarcinogen treatments. Survival rates for 41 weeks after carcinogentreatment did not differ from controls.

Rabbit polyclonal antibodies will be raised against human secretorycomponent, which will be obtained from customary commercial sources. Theantibodies will be raised and tested by Western immunoblotting withchemiluminescence detection to confirm specificity and species crossreactivity. The antibodies will be immunoaffinity purified. To measurerat IgA and IgM in serum, tissue extracts or secretion samples,radioimmunoassay (RIA) will be used with antibodies purchased fromZymed. Iodine labeling of IgA, IgM and secretory component will be doneby standard methods. A non-radioactive ELISA will also be evaluated tomeasure IgA, IgM and secretory component. The concentrations of IgA andIgM in secretions can also be estimated by Western analysis withdensitometry, according to well known procedures. Secretory componentwill be measured by RIA. RIA/ELISA data will be analyzed by computerusing logit transformations and regression analysis, as in known bythose skilled in the art.

Purified rat plasma IgA, IgM and bulk IgG will be purchased initiallyfrom Zymed. Human sIgA arid human plasma dimeric/polymeric IgA will bepurchased from Accurate Chemicals. As larger supplies become necessaryfor animal tests, plasma IgA and IgM can be purified by the preferredmethods described herein, and sIgA from colostrum. Alternatively,another purification method could be substituted, provided that ityields IgA and IgM preparations with cell growth inhibitory activitycharacteristics and purity at least equal to those described in thepresent cell growth assays.

The environmental carcinogens to be tested will be B[a]P, 1-NP and PhIP.They will be compared to a trioctanoin vehicle control. Tumors appear inresponse to B[a]P, PhIP and 1-NP at 5, 9 and 17 weeks, respectively. Thecarcinogens will be administered for eight weeks at a dose of 50μmol/rat/week. Body weight versus time will be measured. A repeatedmeasures analysis of variance (ANOVA) will be employed to determineoverall group differences in weight. Pair-wise, repeated-measuresanalysis will be employed to determine where differences occur.Cumulative mortality will be measured. The probability of survival willbe evaluated by life-table analysis with death as the end point. Thestatistical difference between pairs of groups will be evaluated by thelog-rank test. Tumor incidence will be evaluated by life-table analysiswith time of first appearance of tumor as the end point. Over theplanned duration of these experiments, the rate of spontaneous mammarytumors is not significant.

For the quantification of mammary gland development, radioisotopelabeling of DNA and estimation of numbers of tumors, applying welldescribed methods. Both right and left cervical, thoracic, abdominal andinguinal glands will be analyzed. Left glands will be fixed for wholemount estimates of the numbers of terminal end buds (TEB), terminalducts (TD) and alveolar buds (AB) structures. Carcinogenicity correlateswith the densities of TEB and TD. The effects of IgA and IgM andcarcinogens will be monitored on these structures as well as on L.I.(Labeling Index) and the numbers of tumors. The right glands will belongitudinally sectioned for autoradiography (i.e., L.I. measurements)and stained for tumor quantification. The scoring of tumors will be doneby three methods. Palpable tumors will be measured, the number of tumorsin whole mounts will be estimated by stereomicroscopy and microtumorswill be counted in the sections. The dose and timing of methyl tritiumlabeled thymidine (³H-TdR) treatment of the animals has been defined.DNA synthesis can be measured at any time prepubertal rats because theestrus cycle has not begun. After puberty, DNA synthesis is measured atestrus. Five rats will be included in each time point. This sample sizehas yielded significant (P<0.05) differences between prepubertal animalsand those at 110 days. The unpaired t test will be used to compare theresults from different age groups to determine when a significantdifference in DNA synthesis has been identified (i.e. P<0.05). The agegroups to be studied will be 30 to 35 days, 35 to 40 days, 40 to 45days, 45 to 50 days, 60 to 65 days, 80 to 85 days, 100 to 110 days, 120to 150 days, 200 to 230 days and retired breeders at 270+ days.

First, the age of young female rats will be identified in which DNAsynthesis is maximized. DNA synthesis will be monitored by ³H-TdRincorporation. This initial study is expected to confirm, under thepresent test conditions, those data reported by others in theliterature. Age groups spanning 20 days to 270+ days will be assessed.When the period of maximum DNA synthesis is identified, IgA and IgMinjections will be used to suppress DNA synthesis during this time.After the period of most rapid DNA synthesis has been identified, thefemales of that group will be treated i.p. with IgA and IgM. Todetermine dose, RIA of the serum collected from each animal group listedabove will be performed to establish the concentrations of IgA and IgMin the circulation of sexually mature adult and multiparous females.After an effective immunoglobulin dose is found, the appropriate agegroup will be treated with IgA/IgM and the effects on carcinogenesisassessed versus control animals. The doses of the immunoglobulins willbe increased until blood levels in the adolescent rat equal or exceedthose of mature females. These doses will be administered before thestart of DNA synthesis and throughout the period of carcinogentreatment. When DNA synthesis has been suppressed as judged by totallabel incorporation into DNA, measurement of L.I. and TEB measurements,the three environmental carcinogens will be administered to separategroups of fifteen rats and monitor tumor development as described above.The unpaired t test will be used to compare the results from between thecontrol group (vehicle only) and each carcinogen treated group. Thedifferences between carcinogen groups will be compared as describedabove. A significant (p<0.05) suppression of carcinogenesis and asignificant suppression of TEB development are expected to beidentified. The expected result is that carcinogens will be lesseffective in those rats receiving DNA synthesis inhibiting doses ofIgA/IgM.

Next, the conditions for inducing increased B-cell populations in breasttissue will be identified. Initially, the B-cell content of mammarytissue as a function of age will be monitored. This control study willbe correlated with the time period of maximum DNA synthesis. The contentof B-cells is expected to be low in those age groups showing a maximumDNA synthesis rate. Next, using oral challenges, the most effective“immunogen” to induce an increased population of B-cells in mammarytissue will be determined. The end point of these studies will be toinduce sufficient numbers of B-cells to prevent the “window” increase inDNA synthesis. When conditions have been established to prevent thisrise, the animal will be treated with carcinogens and monitored fortumor development and survival. The oral “immunization” is expected toreduce the effectiveness of the carcinogen.

All secretory tissues from human adults contain substantial numbers ofIgA and IgM producing immunocytes. The immunocytes in lactating humanmammary are about 0.80% IgA secreting and 10% IgM. We will use theanimal groups described above to evaluate the effect of age on IgA & IgMimmunocytes in rat mammary glands. Immunocytes in histological sectionswill be detected by fluorescence after incubation with secretorycomponent and the appropriate primary and secondary antisera. Detaileddescriptions of the fixation and detection methods have been presented.It is expected that adolescent females will have lower numbers of IgAand IgM immunocytes (p<0.05) than adults or multiparous females.Comparisons between the groups will be based on median values and theMann-Whitney non-parametric test (one tail).

Next, “oral challenges” will be used to increase the numbers of IgA andIgM immunocytes in the mammary glands of immature/pubertal female rats.In contrast to historical suggestions of oral immunization of mucosaltissues, including applications to neoplasia, the present,non-conventional “oral immunization” project preferably includes the useof immunogens that show promise with regard to breast. The mostpromising of these are non-pathogenic strains of E. coli. The first ofthese is E. coli 083 that has been used in humans to increase sIgAsecretions in colostrum. Remarkably, high levels of sIgA were induced incolostrum without causing intestinal disturbances. Ingestion by infantsor non-pregnant adults was without symptoms. The colostrum containednumerous immunocytes that secreted IgA against the O antigen of thebacteria. Furthermore, eight, or more prevalent types of E. coli inducedmilk antibodies/immunocytes against the lipopolysaccharide (LPS) of thebacteria. Indeed, even the LPS alone induced high levels of colostrumimmunocytes secreting IgA. The present study will begin with E. coli 083and the LPS from it. The methods of analysis of antibodies in the bloodand in rat colostrum will be done as described. Dosing of the bacteriumand LPS will be developed to block the “window” of DNA synthesis. Wheneffective dosing regiments have been found, we will analyze the effectsof the carcinogens to determine if they are effective when DNA synthesisis suppressed. Also, the state of differentiation of the gland will beanalyzed by measuring terminal end buds (TEB), terminal ducts (TD) andalveolar buds (AB). Both carcinogenesis and differentiation are expectedto be inhibited.

In a third phase of the studies, it will be determined if disruption ofthe function of the secretory immune system causes adult and multiparousfemale rats to become more sensitive to carcinogens. Virgin female ratsof 114 days or older will be studied as will retired breeders of morethan 250 days age. These animals will be treated with antibody againstthe poly-Ig receptor. The doses of antiserum to disrupt the secretoryimmune system will be established by monitoring IgA/IgM secretion intobile, uterine fluids and breast milk. Also, mammary DNA synthesis willbe monitored. When secretion is blocked effectively, the susceptibilityof these animals to carcinogens will be measured. The disruption of theinteraction of IgA/IgM with the poly-Ig receptor is expected to increaseDNA synthesis in the mammary gland and therefore increase susceptibilityto carcinogens.

Because rats do not undergo menopause, a different approach toinvestigating the possible “window” in mid-life females will be used.For this study, the interaction of IgA and IgM with the poly-Ig receptorwill be disrupted using polyclonal antibodies against the receptor. Theantibodies will be confirmed effective by blocking ¹²⁵I-IgA binding tobreast cancer cell receptors using methods. The effect of theseantibodies in vivo will be measured by monitoring DNA synthesis in theadults of 110 to 120 days, 200 to 220 days and retired breeders. Also,the secretion of IgA, IgM, secretory component and J chain into bile,uterine fluids and breast milk will be monitored by the methodsdescribed. Similar methods with J chain polyclonal antibodies haveproven very effective in rats. When the secretions have been diminishedsatisfactorily, antibody treated animals will be treated simultaneouslywith carcinogens. It is expected that DNA synthesis will increase inadult and multiparous animals treated with the antibodies and that thecarcinogens will become more effective.

Applicability to Humans. Human female breast cancer incidence ratesincrease dramatically after age 50 and now approach one in eight by age75. The existing data suggest that the causal mutations most likelyoccur at earlier ages. However, milk/breast secretions decreasedramatically after menopause. Perimenopausal and postmenopausal womenmay also have a previously unrecognized “window” of increasedvulnerability because the activity of the secretory immune systemdecreases with the approach of mid-life. Accordingly, the IgA, IgM andIgG1 inhibitor compositions will also be employed to aid in determiningwhether mutations can arise later in life due to the natural age relatedreduction in the growth inhibitory function of the secretory immunesystem.

Example 41 Bacterial Oncogenesis and Prevention by Oral Immunization

The present example addresses the cause of breast and prostate cancer,as well as cancers of other steroid hormone responsive tissues, from theperspective of determining what is causing the normal mucosal epithelialcells of these tissues to become transformed to the malignant state. Itis now proposed that certain bacteria are carcinogenic (oncogenic),especially in mucosal epithelial tissues, and a screening procedure forisolating and identifying oncogenic bacteria, or bacterial that arelikely to be oncogenic has been devised.

Also presented herein is a two-fold immunization plan to prevent orreduce the risk of occurrence of cancer of the breast, prostate, andother steroid hormone responsive mucosal endothelial tissues. The firstkind of immunization involves immunizing an individual in theconventional way to invoke a natural immune response in whichantibacterial immunoglobulins target and eliminate specific oncogenicbacteria. The second kind of “immunization,” which was previouslyunknown, is to stimulate the natural secretory immune system to producesteroid hormone reversible cell growth inhibitors (i.e., “immunoglobulininhibitors”), which the inventor has discovered are active forms of IgA,IgM and IgG1. These inhibitors have activity for regulating steroidhormone reversible cell growth in mucosal epithelial tissues, such asbreast and prostate. Alternatively, the individual may be “passivelyimmunized” by local or systemic administration of IgA, IgM and IgG1. Bymeans of their cell growth regulatory function, the active forms of IgA,IgM and IgG1 are believed by the inventor to protect the mucosalepithelial tissues from the deleterious effects of bacterial oncogenesiswhich lead to cancerous cell growth.

As disclosed hereinabove and in U.S. Pat. No. ______ (Atty. Dkt. No.1944-00201)/PCT/US2001/______ (Atty. Dkt. No. 1944-00202) entitled“Compositions and Methods for Demonstrating Secretory Immune SystemRegulation of Steroid Hormone Responsive Cancer Cell Growth,” herebyincorporated herein by reference, the secretory immune systemimmunoglobulins IgA, IgM and IgG1 are potent inhibitors of steroidhormone responsive cancer cell growth in chemically defined serum-freeMedium. This serum-free cell culture system constitutes a preferred invitro model of in vivo tumor cell growth that is superior to previouslyavailable serum-free systems. The inhibitory activity is mediated bypoly-Ig receptor or a poly-Ig-like receptor. Among other things, thisdiscovery has strong physiological significance in humans related to thewell-known production of IgA, IgM and IgG1 in breast tissue and thesecretion of these same immunoglobulins into breast milk. In the past,the IgA, IgM and IgG1 of milk were thought to serve only as anantibacterial protection for the suckling offspring. These sameimmunoglobulins, particularly in the form of polymeric IgA andpentameric IgM and IgG1, may also protect the mother and provide a newmeans of preventing or reducing her risk of breast cancer. Similarnegative regulation by IgA, IgM and IgG1 has also been demonstrated bythe inventor in androgen responsive prostate cancer cells. Analogousresults are also indicated in steroid hormone responsive cancers of allother mucosal epithelial tissues that either secrete or are bathed byIgA, IgM and IgG1 in the body. These include not only tissues of thebreast, prostate, pituitary and kidney, but also any other tissue thatlines a cavity or secretes IgA/IgM/IgG1, such as tissues of thegastrointestinal tract (i.e. oral cavity mucosa, salivary/parotidglands, esophagus, stomach, small intestine and colon), tear ducts andnasal passages, liver and bile ducts, bladder, pancreas, adrenals,kidney tubules and glomeruli, lungs, the female reproductive tract (i.e.ovaries, fallopian tubes, uterus, cervix and vagina) and the secretoryanterior pituitary gland. All of these glandular/mucosal tissues eithersecrete or are bathed by polymeric IgA, secretory IgA (sIgA), IgM andIgG1. Cancers arising from these tissues account for 80% of theepithelial malignancies of humans.

In light of the discovery that the secretory immune systemimmunoglobulins IgA, IgM (and IgG1 in humans) are potent inhibitors ofsteroid hormone responsive cancer cell growth, in the preceding.Example, it has been demonstrated how the steroid hormone responsivetissues in the body may be protected from the cancer causing actions ofcertain environmental carcinogens by enhancement of the IgA, IgM andIgG1 secreted by or coming in contact with those tissues. In this way,DNA synthesis dependent mutations can be prevented or substantiallyreduced in those tissues.

Certain bacterial products, either alone or in cooperation withleukocytes, are responsible for production of “reactive oxygen andnitrogen” that lead to malignant transformation of breast and prostateepithelial cells. Immunity to these oncogenic bacteria can conferresistance to this process and thereby reduce the risk of breast andprostate cancer. By employing the bacterial screening procedures thatare described below, bacteria that are likely to be inducers of cancerin vivo are identified. These bacteria, or a combination of bacteria, orimmunogens derived from the oncogenic bacteria, can then be used todevelop specific antimicrobial therapies. One such antimicrobial therapyincludes the production of secretory immunity via oral administration ofthe inactivated or otherwise attenuated bacteria to confer mucosalimmunity. Alternatively, nasal or rectal administration routes may beemployed to produce mucosal immunity in an individual considered to beat risk of developing cancer in a mucosal tissue. Another means ofprotecting an individual against the oncogenic action of the bacteriaisolated or identified as set forth below is to induce systemic immunityto the bacteria, using conventional techniques for raising systemicantibodies to a microorganism.

Moreover, by employing conventional diagnostic immunology and otherimmune-based tests of plasma or other bodily secretions, it can bedetermined if an individual has been or is actively infected by thesuspected oncogenic bacteria, which has been isolated or identifiedaccording to the screening procedures described below. With thisinformation, predictions can be made as to which individuals may be athigher risk for development of cancer in the affected tissue.Alternatively, or additionally, a variety of conventionalmetabolic/chemical inhibitor approaches may be employed to destroy thepotentially oncogenic bacteria in the affected tissues. For example,administration of an effective dose of an appropriate antibiotic to anindividual infected by an oncogenic bacteria.

In light of the discovery regarding the role of the natural secretoryimmune system in regulating the growth of cancer, and finding indirectsupport in the literature, it is now concluded that bacterial infectionsare likely to be an important factor in development of prostate cancer,and that bacteria are also likely to be a primary cause of cancers ofbreast and other mucosal epithelial tissues. Accordingly, the followingscreening procedures are provided for isolating and identifying bacteriafrom breast and prostate sources and assessing their transformingactivity.

Screening Procedure for Identifying Carcinogenic Bacteria. Human milkwill be collected from pregnant volunteers either directly or viaprofessional organizations working with nursing mothers. Alternatively,nipple aspirate fluid will be obtained from non-cancerous volunteers andbreast cancer patients prior to surgery or chemotherapy, preferably asdescribed (Trock B and McLesky S Proceedings of the Era of Hope 2000Meeting, Department of Defense Breast Cancer Research Program, Atlanta,Ga., June, 2000). Breast tissue samples from non-surgical volunteerswill be collected under conditions that exclude skin origin bacteria.Breast cancer samples obtained during surgery will also be directlycultured and evaluated. Those samples will include normal breast tissue(e.g. from reduction mammoplasty) and tumor specimens from breast cancerpatients.

Specimens of semen/seminal fluid will be obtained from normal volunteersof different ages. Because cancer causing mutations may be present forseveral years before the clinical manifestation of disease, samples willbe collected from young adult men<35 years of age as well as from meninto their seventies (highest rate). In addition, surgical samples willbe cultivated and otherwise analyzed to identify the types of bacteriapresent and their relative frequencies. The samples will be classifiedas (i) bacterial prostatitis, (ii) nonbacterial prostatitis, and (iii)asymptomatic inflammatory prostatitis (Lipsky B A (1999) Am J Med 106,327-334).

Special care will be given to the analysis of clinical samples forbacterial content. Some considerations have been discussed by others(Sandin R L and Rinaldi M (1996) Infect Dis Clin North Am 10, 413-430).Precautions will be taken to avoid inclusion of extraneous bacteria inthe samples, and to ensure quality control, including those indicated.Gram stain negative versus gram stain positive bacteria will beclassified. Gram stain negative bacilli cause most prostatitis (Lipsky BA (1999) Am J Med 106, 327-334). For breast samples, this must still beestablished. This is the first selection process to be used to reducethe number of possible bacteria.

The next selection process will use colony derive bacteria to conductthe “Ames Test” to identify bacteria producing mutagens (Ames B N (1979)Science 204, 587-593). This test is based on the scientifically acceptedconcept that DNA damage appears to be the major cause of cancer. Thisassay employs an in vitro mutagenesis test using the bacteriumSalmonella. The culture medium from each form of bacteria isolated canbe tested directly for mutagenic activity using any of several strainsof Salmonella developed for this purpose. The different types ofscreening methods have been reviewed (Hill D C (1998) Adv Biochem EngBiotechnol 59, 73-121). Addition improvements in the Ames Test have beenintroduced to provide more quantitative evidence that the assay isproviding significant results with respect to cancer bioassays (Bogen KT (1995) Environ Mol Mutagen 25, 37-49). The results will be analyzed bystatistical methods (Kim B S and Margolin B H (1999) Mutat Res 436,113-122). The results of this test will establish which bacterialisolates produce mutagenic metabolites (e.g. reactive oxygen andnitrogen species).

The Ames Test can also be applied to demonstrate that the bacteria causean “oxidative burst” mediated by neurophils and macrophages. In thiscase, the leukocytes are incubated with the bacteria to generate theactive mutagenic species. This approach resolves the issue of whetherthe products of the bacteria are the mutagens themselves or if theactivation of leukocytes is required. The various kinds of mutagenesiswill be considered in light of human oncogenesis criteria (Miller J H(1996) Cancer Surv 28, 141-153).

Another type of selection has special application to breast cancer. Milkcontains the protein lactoferrin (Masson D and Taylor C (1978) J ClinPath 31, 316-327). It is well known to be bactericidal by virtue of itshigh affinity for iron (FeIII) (Arnold R et al. (1977) Science 197,263-265). Most bacteria have an absolute requirement for FeIII to grow.However, some have developed “lactoferrin receptors” that permit them toacquire the necessary iron even through it is in complex withlactoferrin. The inventor predicts that mutagenic types of bacteria inbreast secretions/milk will survive and grow in the presence of highconcentrations of lactoferrin. This offers a potent means of selectingfor the bacteria being sought.

Bacteria that meet the criteria described above will be cultured and themedium tested with non-tumorigenic human breast epithelial cells todetermine if the cells are altered to a malignant phenotype. The test ofaltered growth will first be done in serum-free chemically definedmedium, prepared as described the foregoing examples and in U.S. Pat.No. ______ (Atty. Dkt. No. 1944-00201)/PCT/US2001/______ (Atty. Dkt. No.1944-00202) entitled “Compositions and Methods for DemonstratingSecretory Immune System Regulation of Steroid Hormone Responsive CancerCell Growth”, or in Moreno-Cuevas and Sirbasku et al. (2000b), thedisclosures of which are incorporated herein by reference. Transformedcells have reduced growth factor and adhesion requirements. Also, thecells will be tested for colony formation in standard assays. Normalepithelial cells will not form colonies in soft agar. Tumor ortransformed cells will form colonies. There is a very strong correlationbetween colony forming activity in soft agar and tumorgenicity in hostanimals. These tests are expected to confirm that the mutagenic effectsseen with the Ames Test can be translated to transformation of humanbreast cancer cells. Also, normal human prostate epithelial cells areavailable and will be used to perform a similar sequence of studies.

In addition to meeting the foregoing applicable selection criteria, someof the bacteria are expected by the inventor to also possess animmunoglobulin protease activity, i.e., its own “immunoprotective”mechanism. Both seminal fluid and breast secretions contain highconcentrations of IgA. IgA is secreted by prostate and breast epithelialcells. The secreted IgA acts to kill bacteria in these fluids therebyprotecting the tissue. Several types of organisms are known to secreteproteases that cleave the IgA into inactive Fab and Fc components.Examples are Streptococcus pneumoniae (Wani J H et al. (1996) InfectImmun 64, 3967-3974), Haemophilus influenza serotype b (Poulsen K et al.(1989) Infect Immun 57, 3097-3105), Neisseria gonorrhoeae (Simpson D Aet al. (1988) J Bacteriol 170, 1866-1873), Bacteroides melaminogenicus(Mortensen S B and Kilian M (1984) Infect Immun 45, 550-557). These areonly a few examples of bacterial protease activities that have beendescribed in the literature and which are consistent with, and provideindirect support for, this oncogenic bacterial selection criterion.

Finally, to define the bacteria identity, the inventor will apply PCRmethods (Wagar E A (1996) J Clin Lab Anal 10, 331-334). Techniques thatmay be applied include, for example, (1) use of specific PCR primers forknown and new bacteria, (2) PCR amplification of conserved 16S rRNAsequences, and (3) RDA-PCR which is also called “reverse PCR”. Thistechnique can be used to identify unique infectious agents in diseasetissues. Additional PCR technology is available for most of the microbesthat are likely to be encountered.

Although the foregoing protocol has been described with respect tobreast and prostate fluids and tissue specimens, it should be understoodthat similar protocols can be employed with fluids, secretions or tissuespecimens from other mucosal epithelial tissues, including those of thegastrointestinal tract (i.e. oral cavity mucosa, salivary/parotidglands, esophagus, stomach, small intestine and colon), tear ducts andnasal passages, liver and bile ducts, bladder, pancreas, adrenals,kidney tubules and glomeruli, lungs, the female reproductive tract (i.e.ovaries, fallopian tubes, uterus, cervix and vagina) and the secretoryanterior pituitary gland.

Reduction of Breast Cancer Risk by Immunization. One very importantapplication of the bacteria that are identified as oncogenic, or likelyto cause cancer in breast tissue, is to use oral challenges to developmucosal immunity against the bacteria. For the purposes of thisdisclosure, the term “oncogenic bacteria” refers to the forms ofbacteria that cause cancer. According to a preferred regime forpreventing or reducing the risk of breast cancer, oral immunization willbe administered to men and women of all ages to stimulate the naturalsecretory immune system to produce increased local tissue antibacterialimmunoglobulins IgA and IgM. Existing techniques will be employed, suchas those described (Del Giudice G et al. (1999) Methods 19, 148-155).Because of their established natural antimicrobial properties (HeremansJ F (1970) In: Immunoglobulins, Biological Aspects and Clinical Uses,Merler E, ed. National Academy of Sciences, Washington, D.C., 1970), theinventor expects that the secretory immunoglobulins will prevent orsubstantially reduce the risk of breast and prostate cancer by targetingand eliminating the bacteria. The first phase of this disclosure (i.e.identification of oncogenic bacteria) is a first step toward achievementof the second phase, i.e., implementing natural immune system preventionmethods. Such a prevention method is applicable to reducing the risk ofbreast cancer in women without regard to age, race, existing riskfactors, ethnic background or socio-economic status. Similarly, butpreferably using oncogenic bacteria identified in prostate tissue, thenatural secretory immune system will be stimulated to protect againstprostate cancer in men. In a preferred embodiments, the naturalsecretory immune system will be stimulated to eliminate the oncogenicbacteria via conventional antigen-antibody recognition chemistry, and/orto protect breast and prostate tissue from the deleterious effects ofbacterial oncogenesis via the non-conventional cell growth inhibitoryeffects of the secretory immune system.

B cells of the lamina propria of breast and prostate tissue secrete IgAand IgM. These cells originate from the Peyer's patches of the smallintestine (Owen R L (1999) Seminar Immunol 11, 157-163) and migrate tobreast and prostate after a maturation process in the circulation. Entryof B cells into the circulation is stimulated by oral agents (Boyaka P Net al. (1999) Am J Trop Med Hyg 60 (4 suppl), 35-45). This includesbacterial challenge. The IgA and IgM produced in breast tissue isdestined for secretion into milk (Nathavitharana K A et al. (1995) ArchDis Chil Fetal Neonatal Ed 72, F102-F106). The IgA and IgM produced inprostate tissue are destined for secretion into seminal fluid (Stem J Eet al. (1992) J Reprod Immunol 22, 73-85). The immunoglobulins aretransported across mucosal epithelium by poly-Ig receptor mediatedtranscytosis (Mostov K E (1994) Annu Rev Immunol 12, 63-84). In allsecretions of mucosal tissues, IgA and IgM are primary antimicrobialagents. This process has been described in detail (Mestecky J and McGheeJ R (1987) Adv Immunol 40, 153-245).

After identifying the types and strains of bacteria most likely to causebreast and prostate cancer, inactivated forms or attenuated forms of thebacteria will be used as oral challenges to develop mucosal immunityusing known techniques such as those described (Viret J F et al. (1999)Infect Immun 67, 3680-3685). A similar approach was used by Sabin todevelop mucosal immunity against the poliovirus (Valtanen S et al.(2000) J Infect Dis 182, 1-5; Fiore L et al. (1997) J Virol 71,6905-6912).

Although oral immunization against breast cancer has been describedabove, it should be understood that protection against cancers of theprostate or other mucosal epithelial tissues, including those of thegastrointestinal tract (i.e. oral cavity mucosa, salivary/parotidglands, esophagus, stomach, small intestine and colon), tear ducts andnasal passages, liver and bile ducts, bladder, pancreas, adrenals,kidney tubules and glomeruli, lungs, the female reproductive tract (i.e.ovaries, fallopian tubes, uterus, cervix and vagina) and the secretoryanterior pituitary gland, may be achieved similarly.

In addition to inducing a conventional type of mucosal immunity againstthe oncogenic bacteria, a second kind of “immunization,” will also beemployed in which the natural secretory immune system is stimulated toproduce active forms of IgA, IgM and IgG1 that have activity forregulating cancer cell growth in mucosal epithelial tissues, especiallythe steroid hormone responsive tissues of breast and prostate.Alternatively, an individual may be “passively immunized” by local orsystemic administration of IgA, IgM and IgG1 to inhibit cancer cellgrowth. As a result of their cell growth regulatory function, the activeforms of IgA, IgM and IgG1 are expected to protect those types oftissues from the deleterious effects of bacterial oncogenesis that leadto cancerous cell growth. For example, by reducing the “imprinting” ofcancer related genetic changes in prepubescent females or by preventinggrowth of early stage tumors in postmenopausal women. Preferably theIgA, IgM and IgG1 are raised against specific oncogenic bacteria,however a broad spectrum of IgA, IgM and IgG1 molecular species appearto exert steroid hormone reversible growth inhibition in these tissues.Continuing studies are directed at pinpointing the most effectivespecies of IgA, IgM and IgG1 for inhibiting cancer cell growth arisingfrom a given tissue and suppressing the effects of transformation.

Conclusions. A bacterial origin for certain cancers is consistent with,and indirectly supported by the work of others relating to the possibleinvolvement of bacteria with Hodgkin's disease. Because manyreproductive, child bearing and socio-economic patterns are shared asrisk factors for breast cancer and Hodgkin's disease in young women,there may well be a common etiology. Moreover, in light of the fact thatno viral origin of human breast cancer has been established to date, theinventor concludes that other more common infective agents are morelikely the cause of breast and prostate cancer. One published study hasused Cytomegalovirus (CMV) infection distribution as a surrogate to testthe hypothesis that breast cancer may be of infectious origin(Richardson A (1997) Med Hypotheses 48, 491-497). Although it is notlikely that CMV is causative for breast cancer, it is found in humanmilk and is transmitted to offspring during the breast-feeding period(Diosi P (1997) Roum Arch Microbiol Immunol 56, 165-178). Those reports,viewed in light of the present disclosure, support the inventor'salternate interpretation of that information, i.e., that fortuitousbacterial infection, which spreads like viral infections, is actuallythe origin of breast cancer.

Human milk contains many microorganisms/bacteria. To date, none havebeen identified that are primary candidates as causative agents ofbreast cancer. The published work pertaining to milk microbiology willbe of great benefit when reevaluated by the appropriate discriminatorsof the present screening procedure for identifying oncogenic bacteria.The existing literature contains many candidates that will be examinedas primary causative agents for breast cancer, employing the screeningprocess described herein.

The “reactive outbursts” from bacterial-challenged leukocytes may serveas an additional cause of cancers of the male reproductive tract,including those of prostate. Although this proposal has not beenpresented before regarding a mechanism for the development of prostatecancers, it is consistent with the results of studies reported in theliterature that neutrophil and macrophage overproduction of reactiveoxygen species damage the tissues and sperm (Ochsendorf F R (1999) HumanReprod Update 5, 399-420). Because prostate cancer increasesdramatically with age, one focus of the inventor's furtherinvestigations will be on microorganisms that are common to men over 35years of age. A previous study by others has shown that theEnterobacteriaceae are more frequently involved in prostatitis in thisage group than in younger men (Joly-Guillou M L and Lasry S (1999) Drugs57, 743-750). The Enterobacteriaceae, as well as other potentiallyoncogenic bacteria, will be examined in ongoing studies as primarycausative agents for breast cancer, employing the new screening process.

The secretory immune system is an integral part of the physiology of allmucosal epithelial tissues. Most mucosal tissues secrete immunoglobulins(IgA and IgM) into the lumen of biological passageways. Although breast,prostate, pituitary and kidney cancer cells were employed in theforegoing examples, it should be understood that any tissue that lines acavity and/or secretes IgA/IgM is a candidate for the same or similarcompositions and methods for the diagnosis, treatment, deterrence orprevention. These include the gastrointestinal tract (i.e. oral cavitymucosa, salivary/parotid glands, esophagus, stomach, small intestine andcolon), tear ducts and nasal passages, liver and bile ducts, bladder,pancreas, adrenals, kidney tubules and glomeruli, lungs, the femalereproductive tract (i.e. ovaries, fallopian tubes, uterus, cervix andvagina) and the secretory anterior pituitary gland. All of theseglandular/mucosal tissues either secrete or are bathed by polymeric IgA,secretory IgA (sIgA) and IgM. Cancers arising from these tissues accountfor 80% of the epithelial malignancies of humans.

As discussed in Example 37, it is interesting to note that in ataxiatelangectasia (A-T) there is an increased incidence of malignancies,with epithelial carcinomas being the predominate kind. Laboratoryevaluations of A-T patients also show, among other abnormalities thatabout 75% of the patients are deficient in IgA and IgM. A number ofstudies have indicated that female relatives of A-T patients sufferexcess risk of breast cancer (Easton D F (1994) Int. J. Radiat. Biol 66(6 Suppl), S177-S182) or gastric cancer (J. O. Bay et al. (1998) Int. J.Oncol. 12, 1385-1390). The contribution of heterozygous A-T mutations tofamilial breast cancer is believed not to significant (Chen J et al.(1998) Cancer Res. 58, 1376-1379).

Prior to the present invention, the ability to arrest cell proliferationof early, steroid hormone responsive mucosallepithelial malignancies hasnever been attributed to IgA/IgM/IgG1. In addition to looking at certainbacteria as potential causes of malignancy, exposure to non-pathogenicbacteria may serve to continuously stimulate the body's production ofprotective levels of IgA/IgM/IgG1 to protect against, or counteract, thecell proliferation-causing effects of the harmful bacteria.

Example 42 Treatment of Steroid Hormone Responsive Breast or ProstateCancer by Administration of IgA/IgM/IgG1

In this example it is demonstrated that prolonged inhibition of cancercell growth by IgA/IgM causes cell death. This effect is exploited intherapeutic methods that have been devised.

In the in vitro assays described in preceding Examples, which areconsidered to be model systems for predicting in vivo tumor growtheffects, IgA and IgM were shown to behave as steroid hormone reversibleinhibitors of ER⁺ breast and AR⁺ prostate cancer cell growth in theclassical sense of the long sought after chalones. During the initialstages, the immunoglobulins arrest cell growth without causing celldeath. Steroid hormones can reverse the inhibition during this period.However, as with most cancer cells, prolonged blockage of the cell cyclecauses cell death (this is the well known basis for chemotherapy).Accordingly, these in vitro studies are the basis for the in vivotherapeutic use of IgM in rats to block the growth of carcinogen-inducedmammary tumors and to treat existing tumors after induction, or thosearising from implantation of MTW9/PL2 cells. In contrast to classicalchemotherapy that attacks cells of many different types, the effects ofIgA and IgM are specific for mucous epithelial cells and are non-toxicto normal organs.

Preferably the most active forms and/or subtypes of IgA/IgM/IgG1 will beemployed, i.e., those forms of immunoglobulins that act as negativegrowth regulators for human breast and prostate cancer cells. Thesubclasses and pertinent allotypes of IgA will be investigated forgrowth regulating activity with human breast and prostate cancer cellsin serum-free defined culture and for specific binding of ¹²⁵I-labeledimmunoglobulin to cell membrane receptors. The presently disclosedresults strongly imply that polymeric forms are the primary or onlybiologically active immunoglobulins. To establish this conclusively, theeffects of monomeric, dimeric and polymeric IgA on the growth of the ER⁺human breast cancer cell lines T47D, MCF-7 and ZR-75-1 will be assessed,employing the above-described growth assay procedures and materials.Cell numbers will be determined with triplicate dishes and the resultsconverted to cell population doublings (CPD) to allow a directcomparison of the specific activities of each IgA form. Each form of IgAor fragment will be ¹²⁵I-labeled by the chloramine T method. The labeledforms will be used to assess specific binding as total binding minusbinding in the presence of a 100-fold excess of the same unlabeledpreparation. For each fragment or protein (i.e. those that mediateestrogen effects), the time, concentration and temperature dependence ofbinding will be assessed. Scatchard analysis will be used to estimatethe numbers of sites per cell and association constants (Ka). Reciprocalcompetitions with unlabeled and labeled dimeric IgA will be used toconfirm that the purified types or fragments associate with the samereceptors.

The IgA1 and IgA2 will be purified from serum and human colostrum asdescribed. The monomeric, dimeric and polymeric forms of each will bepurified by size exclusion and ion exchange FPLC. If IgA2 only possessactivity, it will be further separated into the A2(m)1 and A2(m)₂allotypes. The purifications will be monitored exactly as described Ithe literature. If the most active form is dimeric (and polymeric), itwill be additional strong evidence that the poly-Ig receptor ismediating the growth response. Were the monomers found to havesignificant activity, that will imply that the poly-Ig receptor may notbe the (only) active mediating site. Next, the active IgA will befragmented beginning with IgA protease that cleaves at the classicalhinges. The methods will yield separable Fab fragments from IgA1 as wellas a larger fragment containing the J chain, the secretory component andthe Fc fragments, using standard techniques that are known to those ofskill in the art and which can be readily implemented. Each specifiedfragment will be purified and assayed for growth mediating effects andreceptor binding.

Following the confirmatory studies, preferably the most activeimmunoglobulin species (e.g., dimeric IgA, polymeric IgM, and/or IgG1)will be administered to other animal subjects or human patientssuffering from a hormone responsive breast or prostate cancer, or otherglandular/mucosal epithelial cancer. Preferably an effective dosage ofthe immunoglobulin composition will be introduced directly as one ormore intravenous treatments using known methodologies for optimizingdosage and delivering it to the subject. Administration can be doneintraperitoneally or subcutaneously as well. It should not be overlookedthat this treatment protocol is quite different from conventionalimmunotherapies that rely entirely on effecting passive immunity todisease organisms and/or their antigenic determinants. In the presentcase, the treatment is designed to primarily provide the necessary leveland/or forms or subtypes of polymeric/dimeric IgAs, pentameric IgMand/or IgG1 s for binding with the respective poly-Ig and Fcγ receptorson the target cells sufficient to produce the desired inhibition orarrest of cell proliferation.

Formulations and Processes. For introduction into the body,pharmaceutical compositions containing the immunoglobulin inhibitors aremanufactured in a manner that is well known in the art, e.g., by meansof conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping, and lyophilizingprocesses. The physiologically acceptable carriers are non-toxic torecipients at the dosages and concentrations employed. The formulationused varies according to the route of administration selected (e.g.,solution, emulsion, capsule). For solutions or emulsions, suitablecarriers include, for example, aqueous or alcoholic/aqueous solutions,emulsions or suspensions, including saline and buffered media.Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. See, generally, “Remington'sPharmaceutical Science,” 16th Edition, Mack, Ed. (1980). For inhalation,the immunoglobulin inhibitors can be solubilized and loaded into asuitable dispenser for administration (e.g., an atomizer, nebulizer orpressurized aerosol dispenser).

An “effective amount,” as used herein, is defined as that quantity whichalleviates, to any degree, or eliminates the condition for which themammal is being treated. The determination of an effective dose is wellwithin the capability of those skilled in the art. For any composition,the therapeutically effective dose can be estimated initially either incell culture assays (e.g., one of the model assay systems describedherein), or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

The immunoglobulin inhibitors can be administered individually, togetheror in combination with other drugs or agents. For example, ananti-cancer composition is prepared by conjugating a cytotoxic agent ora chemotherapeutic agent to an immunoglobulin inhibitor of steroidhormone responsive cancer cell growth, the inhibitor comprising IgA, IgMor IgG1, or any combination of those.

Example 43 Monoclonal Antibodies That Mimic or Block IgA or IgM Bindingto the Poly-Ig Receptor

In this Example, the use of new monoclonal antibodies that act like IgAand IgM to inhibit breast and prostate cancer growth by binding to thepoly Ig receptor, or that act to block the binding of IgA/IgM to thepoly-Ig receptor is described. Also described is a method of developinghybridoma cells that secrete both mimicking and blocking antibodies. Themonoclonal antibodies will be raised as described (Kohler G and MilsteinC (1975) Nature (Lond) 256, 495).

Mimicking Antibodies. IgA and IgM mimicking monoclonal antibodies willbe used in treatment protocols either alone or with conventionalanti-hormone therapy. They also will be used in diagnostic methods toanalyze patient specimens for poly-Ig receptor content byimmunohistochemistry and other standard immunological techniques thatare well known in the art.

Blocking Antibodies. A second class of monoclonal antibody secretinghybridoma cells will be obtained from the same protocols used togenerate the hybridoma cells that secrete mimicking antibodies. Thissecond type of blocking monoclonal antibodies prevents the binding ofIgA to the poly-Ig receptor. The hybridoma cells that secrete this typeof antibody and the antibodies themselves are useful reagents. Blockingantibodies will have a variety of therapeutic and/or diagnostic uses.

Features. One advantage of using monoclonal antibodies that mimic theIgA/IgM binding to the poly-Ig receptor is that the poly-Ig receptor canbe targeted as a new site for anticancer intervention. Whilecommercially prepared polyclonal antibodies against the receptor areavailable (Accurate Chemicals), there are no reports of theirapplicability to human therapy. It is not likely that rabbit polyclonalantibodies against the receptor will be useful in humans due to thestrong antigenic response they will elicit. Also, there are two reportsof panels of monoclonal antibodies directed against epitopes of IgA(Reimer C B et al. (1989) Immunol Lett 21, 209-216) and IgA plus thereceptor (Mestecky J et al. (1996) J Immunol Methods 193, 103-148). Noneof these monoclonal antibodies has been tested for activity as ananticancer agent, nor is there any evidence that any act on the poly-Igreceptor to either mimic or block the action of IgA on breast orprostate cancer cells or on cancer cells of any of the other IgA or IgMsecreting tissues of the body. One monoclonal antibody, MAB 6G11, hasbeen described that binds to domain 1 of the poly-Ig receptor (Bakos M Aet al. (1994) Molecular Immunology 31, 165-168). This same domain alsobinds IgA and IgM, implying that MAB 6G11 may be a blocking or possiblya mimicking antibody. However, direct studies of this aspect were notreported, and the monoclonal antibody was not used for anti-cancerpurposes.

Examples of other Types of Monoclonal Antibodies to Receptors. Therehave been similar projects based on the receptors for other hormones andgrowth factors. The use of blocking monoclonal antibodies as therapy forcancer is known (Baselga J and Mendelsohn J (1994) PharmacologyTherapeutics 64, 127-154). The best example is the monoclonal antibodiesraised against the epidermal growth factor (EGF) receptor Baselga J andMendelsohn J (1994) Breast Cancer Res Treat 29, 127-138). Anotherexample is the monoclonal antibody rhuMab against the HER2/Neuproto-oncogene receptor which is over expressed by breast cancer cellsBaselga J et al. (1996) J Clin Oncol 14, 737-744). These immunoglobulinsare designed to block the growth stimulating effects of EGF/transforminggrowth factor (TGFα). Both EGF and TGFα cause cancer cell growthincluding breast and prostate. Anti-EGF and anti-HER2/neu receptormonoclonal antibodies are now commercial anticancer products.

The Target is a Negative Acting Receptor. In the case of growth factorreceptor directed antibodies, the growth factor competes for (and oftenneutralizes) the inhibiting action of the immunoglobulin, which can be asignificant problem. In contrast, the presently described mimickingantibodies target a negative acting receptor. The presence of endogenousIgA or IgM has no effect because the monoclonal antibody and the naturalligand have the same function, i.e., they both inhibit growth. There isno need to be concerned about the presence of IgA or IgM, as they willnot interfere with this treatment. The mouse monoclonal antibodiesagainst the poly-Ig receptor will be converted to human immunoglobulinsby genetic engineering. This will prevent an immunological responseagainst the mouse epitopes that will reduce antibody effectiveness.Monoclonal antibody therapy is non-invasive and can be administeredfrequently over a long duration. Both mimicking and blocking monoclonalantibodies are important because both are expected to have therapeuticvalue. The poly-Ig receptor is localized in mucosal tissues (e.g. GItract, lungs, breast ducts, prostate gland, uterine lining, ovary,kidney tubules and urinary tract, and salivary gland) (Brandtzaeg P(1995) Acta Path Microbiol Immunol Scand 103, 1-19). An importantadvantage of this disclosure is that monoclonal antibodies against thebreast/prostate poly-Ig receptor can also be expected to havetherapeutic effects with cancers of at least some of these othertissues.

Development Protocols. Various strategies may be used to raisemonoclonal antibodies to the human poly-Ig receptor. One approach is touse standard solid-phase chemical synthesis to prepare peptidescorresponding to the known amino acid sequence of the extracellulardomain of the poly-Ig receptor. The extracellular ligand-binding domain,which is approximately 80% of the whole receptor, was first named the“secretory component” because it was found in association with secretedIgA and IgM. Monoclonal antibodies against secretory component can beassayed to determine if they act as mimicking or blocking agents,employing a cell growth assay described herein. An alternative approachwill be to use a combination of immunoprecipitation, affinitychromatography and immunoaffinity chromatography to purify the intact(complete) poly-Ig receptor. The purified receptor will then be used toraise monoclonal antibodies, which can then be screened for mimickingand blocking activity in a suitable cell growth assay described above.

Example 44 Delivery of Chemotherapeutic Agents and Cytotoxins to CancerCells via IgA/IgM/IgG1 or Monoclonal Antibodies to Poly-Ig Receptor

In this Example, polymeric IgA/IgM and monoclonal antibodies to thepoly-Ig receptor are used to deliver chemotherapeutic agents andcytotoxins to breast cancer and prostate cancer cells, and thereby causethe cancer cells to die. The specific delivery of cytotoxic agents tocancer cells has a long history (Shimizu N (1987) Methods Enzymol 147,382-387). The conceptual basis of this approach is to chemicallyconjugate a cytotoxic protein or compound to an antibody or hormone thatdelivers the toxin-conjugate to cancer cells specifically, therebycausing their death. Very commonly, these agents are linked tomonoclonal antibodies with (relative) specificity for the type of cancertargeted. The monoclonal antibodies usually are directed against cellsurface receptors for hormones or growth factors or other over expressedcell membrane proteins.

Toxin-IgA/IgM/poly Ig Receptor Conjugates are New. Of the identifiableliterature related to toxin conjugates, approximately 50% appears topertain to cancer related applications of this technology, none of whichrefer to IgA or IgM as vehicles or to the poly-Ig receptor as a targetfor toxin conjugates. Although several extensive reviews of the topichave been published, and many reports have been published on the statusand problems associated with the use of monoclonal antibodies fordiagnosis and treatment of cancer, none of those references describe theuse of IgA, IgM or poly-Ig receptor/toxin conjugates in breast orprostate cancer. Certain toxin conjugates have been previously describedfor breast cancer treatment, including several bifunctional reagents,and fusion proteins between ligands and antibodies and toxins. A rangeof protein and compound toxins are available, and it is envisioned thatone or more of those will be suitable for conjugating to IgA, IgM or thepoly-Ig receptor. A preferred toxic substance for conjugating to IgA,IgM or the poly-Ig receptor topic is an iron-containing compound,suitable for effecting the delivery of Fe (III) to cells, according tothe present method. As demonstrated in previous Examples, Fe (III) is apotent cytotoxin for ER⁺ breast cancer cells and AR⁺ prostate cancercells.

Advantages. Although many different monoclonal antibodies have beendeveloped and used to target both chemical and protein toxins to cancercells, the present approach employs IgA/IgM as the preferred vehicle(s)for delivery of the toxins and is directed toward a specific target(e.g., the poly-Ig receptor). The use of the poly-Ig receptor isadvantageous because that receptor is more localized tosecretory/mucosal epithelial tissues that are the primary origins of themajor cancers of the body than are the other targets that are typicallyused for targeting of toxins to cancer cells. The discovery thatpolymeric IgA and IgM regulate estrogen responsive (ER⁺) breast cancercells and androgen responsive (AR⁺) prostate cancer cells has opened newpossibilities with regard to targeting the receptor that mediates theirfunction. Since non-mucosal cells do not express the poly-Ig receptor,therapeutic methods that target poly-Ig receptor bearing cancer cellsvia the secretory immune system also have certain advantages. Onebenefit of such a method is that many important organs (e.g., heart andbrain) will not be affected by the treatment.

While the preferred embodiments of the invention have been shown anddescribed, modifications thereof can be made by one skilled in the artwithout departing from the spirit and teachings of the invention. Forexample, one of skill in the art can readily appreciate that for manyapplications, such as estimating risk of cancer, establishing aprognosis, diagnosing, treating, or preventing cancer, a number of themethods, strategies, techniques and compositions described in theExamples may in some cases be advantageously combined, or used inconjunction, to provide a desirable test, treatment or composition.Likewise, in some embodiments, it may be desirable to combine one of thenew methods or compositions with a conventional anti-cancer therapy ortest procedure. The embodiments described herein are exemplary only, andare not intended to be limiting. Many variations and modifications ofthe invention disclosed herein are possible and are within the scope ofthe invention. Accordingly, the scope of protection is not limited bythe description set out above, but is only limited by the claims whichfollow, that scope including all equivalents of the subject matter ofthe claims. Each and every claim is incorporated into the specificationas an embodiment of the present invention. Thus the claims are a furtherdescription and are an, addition to the preferred embodiments of thepresent invention. The disclosures of U.S. Provisional PatentApplication Nos. 60/203,314 filed May 10, 2000; 60/208,348 filed May 31,2000; 60/208,111 filed May 31, 2000; 60/229,071 filed Aug. 30, 2000 and60/231,273 filed Sep. 8, 2000, are hereby incorporated herein byreference. All patents, patent applications and publications citedherein are hereby incorporated herein by reference.

1-65. (canceled)
 66. A method to aid in detecting or diagnosing cancerin a mammalian subject comprising: obtaining a population of cells froma mucosal epithelial tissue specimen taken from said subject, anddetermining at least one of a first set of conditions selected from thefollowing: (a) absence of a poly-Ig receptor gene from said cells; or(b) absence of heterozygosity for said poly-Ig receptor gene in saidcells; wherein said absence is measured by comparison to similardeterminations in non-neoplastic cells from said subject and/or thesubject's previous test results, or by comparison to a predeterminedstandard value, and wherein the presence of at least one said conditionsis suggestive or indicative of the presence of a cancerous orprecancerous lesion in said subject, and an absence of one or more ofsaid conditions being suggestive or indicative of the absence of acancerous or precancerous lesion in said subject.
 67. The method ofclaim 66, further comprising determining at least one additionalcondition selected from the following: (a) absence of heterozygosity fora Fcγ receptor gene in said cells; (b) absence of a Fcγ receptor genefrom said cells; (c) absence or diminution of a Fcγ receptor in saidcells; (d) absence of heterozygosity for a TGF-β receptor gene in saidcells; (e) absence of a TGF-β receptor gene from said cells; (f) absenceor diminution of a TGF-β receptor in said cells; or (g) presence orabsence of an estrogen receptor in said cells; wherein said absence ismeasured by comparison to similar determinations in non-neoplastic cellsfrom said subject and/or the subject's previous test results, or bycomparison to a predetermined standard value, and wherein the presenceof at least one said additional conditions is suggestive or indicativeof the presence of a cancerous or precancerous lesion in said subject,and an absence of one or more of said conditions being suggestive orindicative of the absence of a cancerous or precancerous lesion in saidsubject.
 68. The method of claim 66, wherein said first condition isdetermining the absence of a poly-Ig receptor gene from said cells. 69.The method of claim 66, wherein said first condition is determining theabsence of heterozygosity for said poly-Ig receptor gene in said cells.70. The method of claim 67, wherein one additional condition isdetermined.
 71. The method of claim 70, wherein said one additionalcondition is selected from the following: (a) absence of heterozygosityfor a Fcγ receptor gene in said cells; (b) absence of a Fcγ receptorgene from said cells; or (c) absence or diminution of a Fcγ receptor insaid cells.
 72. The method of claim 70, wherein said one additionalcondition is selected from the following: (d) absence of heterozygosityfor a TGF-β receptor gene in said cells; (e) absence of a TGF-β receptorgene from said cells; or (f) absence or diminution of a TGF-β receptorin said cells.
 73. The method of claim 70, wherein said one additionalcondition is: (g) presence or absence of an estrogen receptor in saidcells.
 74. The method of claim 67, wherein at least two additionalconditions are determined.
 75. The method of claim 74, wherein said atleast two additional conditions are selected from the following: atleast one of: (a) absence of heterozygosity for a Fcγ receptor gene insaid cells; (b) absence of a Fcγ receptor gene from said cells; or (c)absence or diminution of a Fcγ receptor in said cells; and at least oneof: (d) absence of heterozygosity for a TGF-β receptor gene in saidcells; (e) absence of a TGF-β receptor gene from said cells; (f) absenceor diminution of a TGF-β receptor in said cells.
 76. The method of claim74, wherein said at least two additional conditions are selected fromthe following: at least one of: (a) absence of heterozygosity for a Fcγreceptor gene in said cells; (b) absence of a Fcγ receptor gene fromsaid cells; or (c) absence or diminution of a Fcγ receptor in saidcells; and (g) presence or absence of an estrogen receptor in saidcells.
 77. The method of claim 74, wherein said at least two additionalconditions are selected from the following: at least one of: (d) absenceof heterozygosity for a TGF-β receptor gene in said cells; PreliminaryAmendment (e) absence of a TGF-β receptor gene from said cells; or (f)absence or diminution of a TGF-β receptor in said cells; and (g)presence or absence of an estrogen receptor in said cells.
 78. Themethod of claim 74, further comprising determining at least oneadditional condition selected from the following: (a) absence ofheterozygosity for a progesterone receptor in said cells; (b) absence ofa progesterone receptor gene from said cells; (c) absence or diminutionof a progesterone receptor in said cells; (d) absence of heterozygosityfor insulin-like growth factor-I, or a receptor therefor, in said cells;(e) absence of an insulin-like growth factor-I gene, or a receptortherefor, from said cells; (f) absence or diminution of an insulin-likegrowth factor I, or a receptor therefor, in said cells; (g) absence ofheterozygosity for an epidermal growth factor, or a receptor therefor,in said cells; (h) absence of an epidermal growth factor gene, or areceptor therefor, from said cells; (i) absence or diminution of anepidermal growth factor, or a receptor therefor, in said cells; (j)absence of heterozygosity for a fibroblast growth factor, or a receptortherefor, in said cells; (k) absence of a fibroblast growth factor gene,or a receptor therefor, from said cells; (l) absence or diminution of afibroblast growth factor, or a receptor therefor, in said cells; whereinsaid absence is measured by comparison to similar determinations innon-neoplastic cells from said subject and/or the subject's previoustest results, or by comparison to a predetermined standard value, andwherein the presence of at least one said additional conditions issuggestive or indicative of the presence of a cancerous or precancerouslesion in said subject, and an absence of one or more of said conditionsbeing suggestive or indicative of the absence of a cancerous orprecancerous lesion in said subject.
 79. The method of claim 74, whereinthree additional conditions are determined.
 80. The method of claim 79,wherein said three additional conditions are selected from thefollowing: at least one of: (a) absence of heterozygosity for a Fcγreceptor gene in said cells; (b) absence of a Fcγ receptor gene fromsaid cells; or (c) absence or diminution of a Fcγ receptor in saidcells; and at least one of: (d) absence of heterozygosity for a TGF-βreceptor gene in said cells; (e) absence of a TGF-β receptor gene fromsaid cells; or (f) absence or diminution of a TGF-β receptor in saidcells; and (g) presence or absence of an estrogen receptor in saidcells.
 81. The method of claim 66 or 67, wherein the cancerous orprecancerous lesion is associated with breast cancer, prostate cancer,or colon cancer.
 82. The method of claim 81, wherein said cancerous orprecancerous lesion is associated with breast cancer.